Protein scaffolds for disordered regions

Abstract

The present disclosure provides proteins that can be used as scaffolds to bind to specific targets, including specific proteins or peptides of interest, and fusion proteins having such scaffolds. Such proteins or peptides of interest may include disordered regions. The present invention also relates to various uses of the scaffolds, including diagnostic agents, therapeutic agents, detection reagents, and vaccines. Protein scaffolds can be tailored to specifically bind to targets using the methods described herein.

Description

[Technical Field]

[0001] cross reference This application claims priority to and benefit of U.S. Provisional Application No. 63 / 379,648, filed October 14, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to protein scaffolds that specifically bind to disordered regions of target proteins, and methods for making, screening, and using such protein scaffolds. [Background technology]

[0003] Protein targeting based on experimentally determined or computer-modeled three-dimensional structures has become one of the fundamental techniques in modern drug discovery. This has led to significant attention being paid to secondary structures (e.g., alpha helices or beta sheets) in order to target interactions with proteins based on these structures. However, although most proteins or their segments fold into defined three-dimensional structures, research over the past several decades has discovered that there are many polypeptide segments that do not fold into defined structures. In fact, some proteins can still perform their functions in an unstructured / disordered state. These disordered regions of target proteins have not been fully utilized in drug development. Therefore, there is a need for target protein discovery that focuses on these disordered regions. The embodiments described herein fulfill this need and provide related advantages. Summary of the Invention

[0004] The present disclosure provides engineered scaffold proteins that bind to specific target proteins or peptides of interest, and fusion proteins comprising such scaffolds, as well as various uses of the engineered scaffold proteins and fusion proteins.

[0005] Provided herein is an engineered scaffold protein capable of specifically binding to a disordered region in a target peptide, the engineered scaffold protein comprising one or more binding domains comprising one or more linking units and one or more hinge units, wherein the one or more linking units are capable of binding to the disordered region of the target peptide and comprise one or more amino acid changes compared to a wild-type (WT) counterpart, and the one or more hinge units are capable of stabilizing the structure of the binding domain for binding, wherein the engineered scaffold protein has increased target peptide binding activity compared to the WT counterpart of the engineered scaffold. Also provided herein is an engineered scaffold protein in which the increased binding activity includes increased binding frequency, binding rate, binding time, binding affinity, or any combination thereof. Also provided herein is an engineered scaffold protein having reduced immunogenicity compared to the corresponding WT, increased solubility compared to the corresponding WT, increased stability compared to the corresponding WT, increased or decreased hydrophobicity compared to the corresponding WT, increased or decreased hydrophilicity compared to the corresponding WT, increased or decreased surface charge compared to the corresponding WT, or any combination of the foregoing. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the irregular region is located in an internal loop, C-terminal tail, or N-terminal tail of the target peptide. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the irregular region in the target peptide comprises a linear epitope bound by a binding unit. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the linear epitope has a length of about 4 to about 40, about 4 to about 30, or about 4 to about 25 amino acids.Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is comprised in a polypeptide, protein, or protein complex. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and the length of one protein in the polypeptide, protein, or protein complex is greater than about 30 amino acids. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and the weight of one protein in the polypeptide, protein, or protein complex is greater than about 100 daltons. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is comprised in an extracellular protein. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is comprised in a membrane protein. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is contained in an extracellular protein or membrane protein, including a receptor, an ion channel, or a secreted protein. Also provided herein is an engineered scaffold protein capable of specifically binding to an irregular region in a target peptide, wherein the target peptide is contained in an extracellular protein, including a GPCR. Also provided herein is an engineered scaffold protein in which the binding unit has an extended structure.Also provided herein are engineered scaffold proteins having a three-dimensional conformation in which, when the binding unit is folded into the three-dimensional conformation, the binding unit comprises one or more amino acids that are antisense to one or more amino acids in a disordered region of a target peptide as determined by sense-antisense amino acid pairing. Also provided herein are engineered scaffold proteins comprising two binding units. Also provided herein are engineered scaffold proteins in which each of the one or more binding units comprises about 40 to about 200 amino acids, about 60 to about 150 amino acids, or about 80 to about 100 amino acids. Also provided herein are engineered scaffold proteins in which each of the one or more binding units comprises one or more helices, including alpha helices, 3.10 helices, and / or pi helices. Also provided herein are engineered scaffold proteins in which each of the one or more binding units comprises one or more alpha helices. Also provided herein are engineered scaffold proteins in which each of the one or more binding units comprises two, three, four, five, six, seven, eight, nine, ten, or more amino acid changes compared to their wild-type counterparts. Also provided herein are engineered scaffold proteins, wherein each of the one or more hinge units has about 40 to about 200 amino acids, about 60 to about 150 amino acids, or about 80 to about 100 amino acids. Also provided herein are engineered scaffold proteins, wherein one or more hinge units have a concave or partially concave structure. Also provided herein are engineered scaffold proteins, wherein one or more hinge units have a partially flexible conformation that can accommodate an irregular region of a target peptide when the irregular region is bound by the engineered scaffold protein. Also provided herein are engineered scaffold proteins, wherein one or more hinge units comprise one or more beta-sheet strands, linear peptides, covalent interactions, non-covalent interactions, chemical agents, or any combination thereof.Also provided herein are engineered scaffold proteins in which the hinge unit comprises about 3 to about 12 beta-sheet protein chains or about 6 to about 10 beta-sheet protein chains forming one or more beta sheets. Also provided herein are engineered scaffold proteins in which the hinge unit comprises one or two beta sheets. Also provided herein are engineered scaffold proteins in which one or more hinge units are covalently attached to one or more linkage units, or are linked by one or more linkage units, or a combination thereof. Also provided herein are engineered scaffold proteins in which one or more linkage units are one or more linkers. Also provided herein are engineered scaffold proteins in which one or more linkers comprise a peptide linker. Also provided herein are engineered scaffold proteins in which one or more linkage units and one or more hinge units are connected as monomers. Also provided herein are engineered scaffold proteins in which one or more hinge units are attached to the N-terminus or C-terminus of one or more linkage units. Also provided herein is an engineered scaffold protein, wherein the engineered scaffold protein comprises two linking units and one hinge unit, wherein the first linking unit is attached to the N-terminus of the hinge unit and the second linking unit is attached to the C-terminus of the hinge unit.Also provided herein is an engineered scaffold protein, wherein the engineered scaffold protein comprises two linking units and one hinge unit, wherein the first linking unit is attached to the N-terminus of the second linking unit and the hinge unit is attached to the C-terminus of the second linking unit.Also provided herein is an engineered scaffold protein, wherein two or more linking units are antiparallel structures.Also provided herein is an engineered scaffold protein, wherein two or more linking units are hemisymmetric.Also provided herein are engineered scaffold proteins in which one or more linking units and one or more hinge units are multimeric, and in the presence of a target peptide, the one or more linking units and one or more hinge units form a binding domain. Also provided herein are engineered scaffold proteins in which the binding domain is derived from any one of the scaffold proteins listed in Table 1. Also provided herein are engineered scaffold proteins in which the binding domain is a binding groove structure. Also provided herein are engineered scaffold proteins in which the binding domain has a clamshell structure. Also provided herein are engineered scaffold proteins further comprising one or more immunoglobulin units. Also provided herein are engineered scaffold proteins comprising two immunoglobulin units. Also provided herein are engineered scaffold proteins in which one or more immunoglobulin units are covalently attached to the binding domain, hinge unit, linking unit, or a combination thereof, or attached via a linking unit. Also provided herein are engineered scaffold proteins that do not comprise an immunoglobulin unit. Also provided herein are engineered scaffold proteins having an isoelectric point of 3.5 to 9, 4 to 8.5, or 4.5 to 8, as measured in an electrophoresis assay. Also provided herein are engineered scaffold proteins whose linking units comprise an amino acid sequence at least 65% identical to any one of the sequences in Table 4. Also provided herein are engineered scaffold proteins whose hinge units comprise an amino acid sequence at least 65% identical to any one of the sequences in Table 5. Also provided herein are engineered scaffold proteins whose disordered region in the target peptide comprises an amino acid sequence selected from the list of sequences of the bound peptides listed in Table 3, or variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes or more.Also provided herein is an engineered scaffold protein, wherein the engineered protein scaffold is conjugated with a heterologous agent to extend the half-life of the engineered scaffold. Also provided herein is an engineered scaffold protein, wherein the heterologous agent is selected from the group consisting of polyethylene glycol (PEG), human serum albumin (HSA), and a variant Fc region of an antibody. Also provided herein is an engineered scaffold protein, wherein the half-life of the engineered protein scaffold is extended compared to its wild-type counterpart. Also provided herein is an engineered scaffold protein, wherein the disordered region in the target peptide comprises an amino acid sequence selected from the list of sequences of the bound peptides listed in Table 7, or variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid changes. Also provided herein is an engineered scaffold protein, further comprising one or more epitope masking units. Also provided herein is an engineered scaffold protein, wherein the epitope masking units interact with the binding units of the engineered scaffold protein. Provided herein are engineered scaffold proteins in which an epitope masking unit is linked to the N-terminus, C-terminus, or mid-loop of the engineered scaffold protein. Also provided herein are engineered scaffold proteins in which an epitope masking unit is linked to the N-terminus, C-terminus, or mid-loop of the engineered scaffold protein. Also provided herein are engineered scaffold proteins in which the epitope masking unit can partially mask the binding unit, support the tertiary structure of the engineered scaffold protein, improve the immunogenicity of the engineered scaffold protein, or any combination thereof. Also provided herein are engineered scaffold proteins in which the epitope masking unit replaces the disordered region of the target peptide when the binding unit binds. Also provided herein are engineered scaffold proteins having a functional classification selected from the list of functional classifications of human protein scaffolds listed in Table 2. Also provided herein are engineered scaffold proteins comprising an amino acid sequence at least 65% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence at least 75% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence at least 80% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence at least 85% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence at least 95% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence at least 97% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence at least 98% identical to any one of the sequences set forth in Table 6.Also provided herein is an engineered scaffold protein comprising an amino acid sequence that is at least 99% identical to any one of the sequences set forth in Table 6. Also provided herein is an engineered scaffold protein comprising an amino acid sequence that is identical to any one of the sequences set forth in Table 6.

[0006] Provided herein is a fusion protein comprising the engineered scaffold provided herein and a fusion partner.Also provided herein is a fusion protein in which the fusion partner is an enzyme.Also provided herein is a fusion protein in which the enzyme catalyzes ubiquitination, post-translational modification, proteolytic cleavage, dephosphorylation, trans-cis isomerization, protein chaperone activity, nucleic acid-modifying protein, ATPase or GTPase activity.Also provided herein is a fusion protein in which the fusion partner selectively binds to a specific region on the target protein.Also provided herein is a fusion protein in which the specific region on the target protein is not a disordered region of the target protein.

[0007] Provided herein is a method for binding to a disordered region of a target protein, the method comprising contacting a target protein comprising the disordered region with an engineered scaffold protein provided herein.

[0008] Provided herein is a method for binding to a disordered region of a target protein, the method comprising contacting a target protein comprising the disordered region with a fusion protein provided herein.

[0009] Provided herein is a method for altering the conformation of a protein that includes a target peptide, the method comprising contacting the target peptide with an engineered scaffold protein provided herein.

[0010] Provided herein is a method for altering the conformation of a protein that includes a target peptide, the method comprising contacting the target peptide with a fusion protein provided herein.

[0011] Provided herein are methods of treating a disease, the methods comprising administering to a subject an effective amount of an engineered scaffold protein provided herein.

[0012] Provided herein are methods of treating a disease, comprising administering to a subject an effective amount of a fusion protein provided herein.

[0013] Provided herein is a method of inducing an immune response in a subject in need thereof, comprising administering to the subject an engineered scaffold protein provided herein.

[0014] Provided herein is a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a fusion protein provided herein.

[0015] Provided herein is a method for detecting a target protein, the method comprising contacting a sample suspected of containing the target protein with at least an engineered scaffold protein provided herein.

[0016] Provided herein is a method for detecting a target protein, the method comprising contacting a sample suspected of containing the target protein with at least a fusion protein provided herein.

[0017] Provided herein is a kit that includes at least an engineered scaffold protein provided herein.

[0018] Provided herein is a kit comprising at least a fusion protein provided herein.

[0019] Provided herein is a device comprising at least an engineered scaffold protein provided herein.

[0020] Provided herein is a device comprising at least a fusion protein provided herein.

[0021] Provided herein is a method for detecting a compound in a sample, the method comprising contacting the sample with an engineered scaffold protein provided herein.

[0022] Provided herein is a method for detecting a compound in a sample, comprising contacting the sample with a fusion protein provided herein.

[0023] Provided herein is a pharmaceutical composition comprising an engineered scaffold protein provided herein and a pharmaceutically acceptable excipient.

[0024] Provided herein is a pharmaceutical composition comprising a fusion protein provided herein and a pharmaceutically acceptable excipient.

[0025] Provided herein are methods for preventing, treating, or maintaining disease in a subject in need thereof by administering an effective amount of a pharmaceutical composition provided herein.

[0026] Provided herein are methods for preventing, treating, or maintaining disease in a subject in need thereof by administering an effective amount of a pharmaceutical composition provided herein.

[0027] Provided herein are isolated nucleic acid molecules that encode the engineered scaffold proteins provided herein.

[0028] Provided herein are isolated nucleic acid molecules that encode the fusion proteins provided herein.

[0029] Provided herein are expression vectors that are operably linked to the nucleic acids provided herein.

[0030] Provided herein are expression vectors that are operably linked to the nucleic acids provided herein.

[0031] Provided herein are host cells comprising the vectors provided herein.

[0032] Provided herein are host cells comprising the vectors provided herein.

[0033] Provided herein are polypeptide display libraries comprising the engineered scaffold proteins provided herein.

[0034] Provided herein is a polypeptide display library comprising the fusion protein provided herein.Also provided herein is a polypeptide display library, in which the engineered scaffold protein is displayed on the surface of a virus or yeast, or as a ribosome or RNA-conjugated protein molecule.Also provided herein is a polypeptide display library, in which the fusion protein is displayed on the surface of a virus or yeast, or as a ribosome or RNA-conjugated protein molecule.

[0035] Provided herein are collections of isolated nucleic acid molecules that encode the libraries provided herein.

[0036] Provided herein is a method for obtaining an engineered scaffold that binds to a target, the method comprising: (a) contacting a target ligand with a library provided herein under conditions that allow the formation of an engineered scaffold protein:target ligand complex; and (b) obtaining from the complex a scaffold that binds to the target ligand.

[0037] Provided herein is a method for obtaining a fusion protein that binds to a target, the method comprising: (a) contacting a target ligand with a fusion protein library provided herein under conditions that allow for the formation of a fusion protein:target ligand complex; and (b) obtaining from the complex a fusion protein that binds to the target ligand.

[0038] Provided herein is a method for obtaining at least two engineered scaffold proteins that bind to a target, the method comprising: (a) contacting a target ligand with the library provided herein under conditions that allow the formation of an engineered scaffold:target ligand complex; (b) binding the complex with a cross-linking agent, wherein cross-linking of the complex induces a detectable response; and (c) obtaining from the complex the engineered scaffold proteins that bind to the target. Also provided herein is a method for obtaining at least two engineered scaffold proteins that bind to a target, wherein the engineered scaffold proteins recognize the same epitope. Also provided herein is a method for obtaining at least two engineered scaffold proteins that bind to a target, wherein the engineered scaffold proteins recognize distinct epitopes. Also provided herein is a method for obtaining at least two engineered scaffold proteins that bind to a target, wherein the cross-linking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag.

[0039] Provided herein is a method for obtaining at least two fusion proteins that bind to a target, the method comprising: (a) contacting a target ligand with the fusion protein library provided herein under conditions that allow the formation of a fusion protein:target ligand complex; (b) binding the complex with a cross-linking agent, wherein cross-linking of the complex induces a detectable response; and (c) obtaining the fusion proteins that bind to the target from the complex. Also provided herein is a method for obtaining at least two fusion proteins that bind to a target, wherein the fusion proteins recognize the same epitope. Also provided herein is a method for obtaining at least two fusion proteins that bind to a target, wherein the fusion proteins recognize different epitopes. Also provided herein is a method for obtaining at least two fusion proteins that bind to a target, wherein the cross-linking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag.

[0040]

[0003] Provided herein is a method for generating an engineered scaffold protein sequence capable of binding to a disordered region of a target peptide, comprising: selecting one or more scaffold protein sequences; evaluating the one or more scaffold protein sequences for one or more desired characteristics, including ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, or any combination thereof; and manipulating the amino acid sequence of the engineered scaffold protein based on the evaluation of the one or more scaffold protein sequences, wherein the engineered scaffold protein is predicted to have enhanced or improved one or more desired characteristics compared to its one or more counterpart scaffold proteins.

[0004] Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to a disordered region of a target peptide, wherein manipulating the amino acid sequence of the engineered scaffold protein comprises manipulating the amino acid sequence to bind to a linear epitope of the target peptide. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to a disordered region of a target peptide, wherein evaluating one or more scaffold protein sequences for one or more desired characteristics comprises weighting one or more factors of the one or more scaffold protein sequences that are associated with the one or more desired characteristics, wherein the one or more weighted factors comprise three-dimensional structure, protein domain(s), amino acid sequence, amino acid charge, amino acid polarity, amino acid hydrophobicity / hydrophilicity, amino acid acidity / basicity, or any combination thereof.Also provided herein is a method for generating engineered scaffold protein sequences capable of binding to disordered regions of target peptides, wherein weighting one or more factors comprises assigning values ​​to one or more weighted factors based on an estimated probability that the weighting factor will improve one or more desired characteristics, assigning values ​​to one or more weighted factors and measuring the deviation of the values ​​from a target value or a value assigned to the factor of a second scaffold protein, or both of the foregoing. Also provided herein is a method for generating engineered scaffold protein sequences capable of binding to disordered regions of target peptides, further comprising predicting whether an engineered amino acid sequence represents an engineered scaffold protein exhibiting improved characteristics, whether one or more changes in one or more weighted factors improve the value compared to a target value or threshold value or a value assigned to such factor of a second scaffold protein, or both of the foregoing. Also provided herein is a method for generating engineered scaffold protein sequences capable of binding to disordered regions of target peptides, wherein the one or more changes comprise one or more amino acid changes. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, wherein the engineered amino acid sequence of the engineered scaffold protein comprises one or more changes compared to a counterpart scaffold protein. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, wherein evaluating one or more scaffold protein sequences comprises manually evaluating the amino acid sequence of one or more scaffold protein sequences or evaluating the amino acid sequence of one or more scaffold protein sequences in a machine learning model.Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, further comprising evaluating another desired characteristic of the engineered amino acid sequence of the engineered scaffold protein, weighting one or more weighted factors of the engineered amino acid sequence of the engineered scaffold protein, or a second or subsequent iteration of both of the foregoing, and providing information for further manipulation of the amino acid sequence of the engineered scaffold protein. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, comprising generating the engineered scaffold protein. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, comprising assaying the engineered scaffold protein. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, wherein the assaying comprises one or more in vitro or in vivo assays. Also provided herein is a method for generating an engineered scaffold protein sequence capable of binding to an irregular region of a target peptide, wherein the assaying comprises in silico simulation and / or machine learning model simulation. Also provided herein are methods for generating sequences of engineered scaffold proteins capable of binding to disordered regions of target peptides, wherein assaying the generated engineered scaffold proteins comprises obtaining data and providing information for further generation of engineered scaffold proteins.

[0041] Provided herein are systems that include instructions capable of implementing the methods provided herein.

[0042] Provided herein are engineered scaffold proteins comprising an amino acid sequence produced by the methods provided herein or the systems provided herein.

[0043] Provided herein are compositions comprising an engineered scaffold protein or a fusion protein provided herein and a therapeutic partner. Also provided herein are compositions in which the therapeutic partner is attached to the engineered scaffold protein or fusion protein, and the therapeutic partner is covalently bound, non-covalently bound, fused, conjugated, and / or linked to the engineered scaffold protein or fusion protein. Also provided herein are compositions in which the therapeutic partner comprises one or more of an anti-cancer drug, an anti-inflammatory drug, an antibacterial drug, an antiviral drug, a cytokine, a toxin, an enzyme, a neuroprotective agent, a soluble factor capture agent, or any combination thereof. Also provided herein are compositions in which the therapeutic partner can be separated from the engineered scaffold protein or fusion protein upon binding to a target peptide. Also provided herein are compositions in which the engineered scaffold protein or fusion protein can be tissue- or cell-specific. Also provided herein are compositions in which the therapeutic partner can be separated from the engineered scaffold protein or fusion protein upon reaching a specific cell or tissue. Provided herein are methods for treating a disease or disorder, comprising administering to a subject in need thereof a composition provided herein.

[0044] Provided herein are methods of chimeric antigen receptor (CAR) T-cell therapy, comprising administering to a subject in need thereof an engineered scaffold protein provided herein, or a fusion protein provided herein.

[0045] Other features and advantages of the invention will be apparent from the detailed description and claims. [Brief explanation of the drawings]

[0046] [Figure 1] Schematic diagrams of engineered scaffolds or their fusion proteins that act as inhibitors for target mutant proteins with disordered regions. (A) The engineered scaffolds or their fusion proteins that highly specifically mask the mutant region and bind to a domain of protein C. (B) An interacting protein, which can be wild-type or mutant, whose interaction causes disease. (C) A protein that, when bound to the disordered region, preferentially interacts with the engineered scaffolds or their fusion proteins (A) and does not cause disease.

[0047] [Figure 2] Schematic diagram of a disease target protein without an engineered scaffold or its fusion protein, and a disease target protein with an engineered scaffold or its fusion protein acting as an inhibitor. The engineered scaffold or its fusion protein (A) highly specifically masks the mutated region (B) and inhibits the interaction.

[0048] [Figure 3] Schematic diagram of an engineered scaffold or its fusion protein (A) that modulates DNA / RNA binding by binding to disordered regions of disease targets, thereby rendering the DNA / RNA unable to bind to the target.

[0049] [Figure 4] Schematic diagrams of a polymer with a disordered region that is incapable of binding to a target and a polymer that is capable of binding to a target after attachment of an engineered scaffold or its fusion protein (A). Binding of the engineered scaffold or its fusion protein to the disordered region modulates DNA / RNA binding.

[0050] [Figure 5]Figure 1 shows a schematic of a fusion protein in which an engineered scaffold is fused to a fusion partner that catalyzes ubiquitination. The scaffold-E3 ligase fusion binds to a disease target containing a disordered region, ubiquitinates the target, and marks it for degradation.

[0051] [Figure 6] Schematic diagrams of fusion proteins in which an engineered scaffold is fused to a fusion partner with a specific enzymatic activity are shown. The engineered scaffold or its fusion protein (A) binds to a disease target with a disordered region, and the enzymatic portion of the fusion protein modulates the biological activity of the target or other proteins in the vicinity of the binding site. Such enzymatic activities include post-translational modification, proteolytic cleavage, dephosphorylation, trans-cis isomerization, protein chaperone activity, catalysis of nucleic acid-modified proteins, ATPase or GTPase activity, etc.

[0052] [Figure 7] Schematic diagrams of a functional protein (left), a misfolded protein with at least one disordered region (middle), and a restored functional protein with two ordered domains (right) after binding of a fusion protein in which two engineered scaffold proteins (A) are fused together or a fusion protein with at least one engineered scaffold protein and a chaperone protein are combined. The functional protein and the resulting restored functional protein are also shown for reference.

[0053] [Figure 8] Schematic of localized protein editing by releasing post-translational modifications (PTMs) in a misfolded protein (center) after binding of a fusion protein (A) comprising an engineered scaffold protein and an enzymatic fusion partner. The functional protein (left) and the resulting restored functional protein (right) are also shown for reference.

[0054] [Figure 9]Schematic of localized protein editing by sequence-specific degradation of misfolded proteins using a fusion protein (A) with an engineered scaffold protein and a degradative fusion partner on the misfolded protein (center). The functional protein (left) and the resulting restored functional protein (right) are also shown for reference.

[0055] [Figure 10] FIG. 1 shows a schematic representation of various forms of engineered scaffold proteins that can be used for therapeutic development, including fusion proteins having an engineered scaffold protein and a fusion partner, PEG-conjugated engineered scaffold proteins, and multispecific scaffold constructs.

[0056] [Figure 11] FIG. 1 shows a diagram of an exemplary process for engineering engineered scaffold proteins.

[0057] [Figure 12] FIG. 1 shows a schematic diagram of an exemplary stepwise optimization process for engineered scaffold proteins.

[0058] [Figure 13] FIG. 1 shows a diagram of an exemplary development pipeline for engineered scaffold proteins.

[0059] [Figure 14] 1 shows a schematic diagram of a fusion protein in which an engineered scaffold is fused to a fusion partner comprising the Fc region of an antibody. The engineered scaffold binds to a target protein with a disordered region expressed on the surface of a target cell, or to a target antigen with a disordered region, and the Fc portion of the fusion protein activates a cascade of events that results in the killing of the target cell and / or the destruction of the target antigen.

[0060] [Figure 15A]A representative SDS PAGE gel of an engineered scaffold protein or its fusion protein migrates through the PAGE gel at the expected size, approximately 22 kDa. The engineered scaffold proteins range in size from approximately 20 kDa to approximately 50 kDa.

[0061] [Figure 15B] Figure 1 shows a size-exclusion chromatogram of the engineered scaffold protein, showing that most of the produced protein passes through the column as monomer (highest peak), with smaller fractions split into dimers and multimeric aggregates. The x-axis represents volume (mL) and the y-axis represents absorbance (Abs, mAu).

[0062] [Figure 16A] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S005, S007, S090) and a no-scaffold control to the target CD74, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0063] [Figure 16B] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S005, S007, S090) and a no-scaffold control to the target KAAG1. The x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0064] [Figure 16C] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S005, S007, S090) and a no-scaffold control to the target CysLT2, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0065] [Figure 16D] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S005, S007, S090) and a no-scaffold control to the target CRACM, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0066] [Figure 16E] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S005, S007, S090) and a no-scaffold control to the target KvI.5, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0067] [Figure 17A] Graph showing improvement in target binding after scaffold optimization, where points represent a no-scaffold control and three engineered scaffold proteins. The x-axis represents target concentration (nM) and the y-axis represents fluorescence (AU).

[0068] [Figure 17B] Figure 1 shows a bar graph depicting the improvement in affinity after scaffold optimization, where each bar represents a no-scaffold control and three engineered scaffold proteins, respectively, and the y-axis represents relative binding.

[0069] [Figure 17C] Figure 1 shows a bar graph depicting the relative binding improvement after scaffold optimization, with each bar representing a no-scaffold control and three engineered scaffold proteins, and the y-axis representing fold change.

[0070] [Figure 17D] 1 shows a bar graph illustrating the improvement in specificity after scaffold optimization. Each bar represents an engineered scaffold protein and the y-axis represents the fold change.

[0071] [Figure 18A] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S058, S065, S090) and a no-scaffold control to the target CD74. The x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0072] [Figure 18B] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S058, S065, S090) and a no-scaffold control to the target KAAG1. The x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0073] [Figure 18C] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S058, S065, S090) and a no-scaffold control to the target CysLT2r, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0074] [Figure 18D] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S058, S065, S090) and a no-scaffold control to the target CRACM, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0075] [Figure 18E] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S058, S065, S090) and a no-scaffold control to the target KvI.5, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0076] [Figure 19A] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S164, S171) and a no-scaffold control to the target CD74, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0077] [Figure 19B] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S164, S171) and a no-scaffold control to the target KAAG1. The x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0078] [Figure 19C] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S164, S171) and a no-scaffold control to the target CysLTR2, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0079] [Figure 19D]1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S164, S171) and a no-scaffold control to the target CRACM, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0080] [Figure 19E] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S164, S171) and a no-scaffold control to the target CRACM, where the x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0081] [Figure 20A] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S177) and no-scaffold controls to the target MERKFMSLQPSISVSEMEPNG (SEQ ID NO: 927). The x-axis represents the concentration of target (nM) and the y-axis represents fluorescence (AU).

[0082] (FIG. 20A) A graph showing the binding affinity of engineered protein scaffolds (S090, S177) and no-scaffold controls to the target MERKFMSLQPSISVSEMEPNG (SEQ ID NO: 927). The x-axis represents the concentration of target (nM) and the y-axis represents fluorescence (AU).

[0083] [Figure 20B] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S177) and no-scaffold control to the target SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928). The x-axis represents the concentration of target (nM) and the y-axis represents fluorescence (AU).

[0084] [Figure 20C] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S090, S177) and no-scaffold control to the target TFSNNNSRNCTIENFKREFFP (SEQ ID NO: 929). The x-axis represents the concentration of target (nM) and the y-axis represents fluorescence (AU).

[0085] [Figure 20D] A diagram of the schematic for identifying epitopes on CYSLTR2 that bind to the engineered scaffold is shown, where the N-terminal binding region was divided into three N-terminally biotinylated peptides (epitope 1 (sequence number 927), epitope 2 (sequence number 929), epitope 3 (sequence number 928)) with a length of approximately 21 AA and an overlap of approximately 10 AA.

[0086] [Figure 21] 1 shows a graph illustrating the improved binding affinity of engineered protein scaffolds (S005, S090, S177) and no-scaffold control to the target CYSLT2R (SEQ ID NO: 928). The x-axis represents the target concentration (nM) and the y-axis represents fluorescence (AU).

[0087] [Figure 22A] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S180, S189, S193, S194) and no-scaffold controls to the target CYSLTR2 epitope (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)). The x-axis represents the concentration of CYSLTR2 peptide (nM), and the y-axis represents fluorescence (AU).

[0088] [Figure 22B] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S180, S189, S193, S194) and no-scaffold controls to the target CYSLTR2 epitope (GMERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 929)). The x-axis represents the concentration of CYSLTR2 peptide (nM), and the y-axis represents fluorescence (AU).

[0089] [Figure 23A] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S193, S194) and no-scaffold controls to the target CYSLTR2 epitope (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)). The x-axis represents the concentration of CYSLTR2 peptide (nM) and the y-axis represents fluorescence (AU).

[0090] [Figure 23B] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S193, S194) and no-scaffold controls to the target KCNA6 epitope (QQQEQQPASGGGGQNGQQAMS (SEQ ID NO: 931)). The x-axis represents the concentration of KCNA6 peptide (nM) and the y-axis represents fluorescence (AU).

[0091] [Figure 23C] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S193, S194) and no-scaffold controls to the target TNFRSF13B epitope (LPPELRRQRSGEVENNSDNSGRYQ (SEQ ID NO: 932)). The x-axis represents the concentration of TNFRSF13B peptide (nM), and the y-axis represents fluorescence (AU).

[0092] [Figure 23D] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S193, S194) and no-scaffold controls to the target CD74 epitope (PPKPVSKMRMATPLLMQALPMGALP (SEQ ID NO: 933)). The x-axis represents the concentration of CD74 peptide (nM) and the y-axis represents fluorescence (AU).

[0093] [Figure 23E] 1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S193, S194) and no-scaffold controls to the target KAAG1 epitope (PGAAAAHLPRWPPPQLAASRREA (SEQ ID NO: 934)). The x-axis represents the concentration of KAAG1 peptide (nM), and the y-axis represents fluorescence (AU).

[0094] [Figure 23F]1 shows a graph depicting the binding affinity of engineered protein scaffolds (S177, S193, S194) and no-scaffold controls to the target CD40LG epitope (NKEETKKENSFEMQKGDQNPQIAAH (SEQ ID NO: 935)). The x-axis represents the concentration of CD40LG peptide (nM) and the y-axis represents fluorescence (AU).

[0095] [Figure 24A] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S070) to the target 18-amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0096] [Figure 24B] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S090) to its target, an 18-amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0097] [Figure 25] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S090) engineered to target an 18-amino acid epitope of CLIP to the target 18-amino acid epitope of CLIP and a 25-amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0098] [Figure 26] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S005) to the target 25 amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0099] [Figure 27A] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S098) to its target, an 18-amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0100] [Figure 27B] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S098) to the target 25 amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0101] [Figure 28] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S058) to its target, an 18-amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0102] [Figure 29A] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S068) to its target, an 18-amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0103] [Figure 29B] Figure 1 shows a graph depicting the binding kinetics of an engineered protein scaffold (S068) to the target 25 amino acid epitope of CLIP. The y-axis is the shift in the interference pattern, which is proportional to the number of CLIP peptide molecules bound by the engineered scaffold (nm). The x-axis is time (seconds).

[0104] [Figure 30A]1 shows a graph depicting the binding affinity of engineered protein scaffolds (v1.0 is S007, v1.1 is S005, v1.2 is S090) and no-scaffold controls to the target GPCR1 (CYSLTR2) (MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP) (SEQ ID NO: 926). The x-axis represents the concentration of GPCR1 peptide (nM) and the y-axis represents fluorescence (AU).

[0105] [Figure 30B] Figure 1 shows a bar graph depicting the relative binding improvement after scaffold optimization, where each bar represents a no-scaffold control and three engineered scaffold proteins (v1.0 is S007, v1.1 is S005, and v1.2 is S090), and the y-axis represents relative binding.

[0106] [Figure 31]

[0023] Figure 1 shows a graph depicting the binding affinity of engineered protein scaffolds (v1.0 is S007, v1.1 is S005, and v1.2 is S090) to two GPCR targets: GPCR1 (CYSLTR2) (MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 926)) and GPCR2 (Urotensin II) (MALTPESPSSFPGLAATGSSVPEPPGGPNATLNSSWASPTEPSSLEDLVATGTI (SEQ ID NO: 972)). The x-axis represents the concentration of GPCR peptide (nM) and the y-axis represents relative fluorescence units (AU).

[0107] [Figure 32] 10 demonstrates the shielding or inhibitory activity of the engineered scaffold proteins described herein. In this figure, after binding by the engineered scaffold proteins provided herein, access to the binding site of CysLTR2 by leukotriene C4 is reduced.

[0108] [Figure 33] 10A-10C demonstrate top and side views of three-dimensional renderings of engineered scaffold proteins with binding groove structures described herein.

[0109] [Figure 34] 10A-10C demonstrate a cross-sectional view of a three-dimensional rendering of an engineered scaffold protein with a binding groove structure as described herein. DETAILED DESCRIPTION OF THE INVENTION

[0110] Disclosed herein are compositions and methods that precisely bind to known sequences of amino acids. Such compositions and methods rely on the engineered scaffold proteins disclosed herein. Also disclosed herein are compositions and methods that bind to disordered regions of various lengths found in target proteins. Such compositions and methods bind to proteins in a sequence-specific manner based on the engineered scaffold proteins. Further disclosed herein are methods for locally, sequence-specifically editing protein structure or function using such engineered scaffold proteins. Further disclosed herein are methods for modulating protein-polymer interaction networks using the engineered scaffold proteins disclosed herein. These engineered scaffold proteins can be used in methods for sensing and detecting biological analytes, inducing local conformational changes, and simultaneously specifically binding to two or more targets by fusing and linking multiple binding moieties together. The compositions and methods described herein can be used to bind to macromolecular targets in the extracellular matrix and intracellular environments. Further disclosed herein are kits, reagents, and instruments that can detect macromolecules in situ using the engineered scaffold proteins described herein. Such kits, reagents, and instruments can be used to detect proteins / peptides, for example, present on a bead or chip surface, for protein fingerprinting, protein sequencing, or protein detection.

[0111] As used in this application, including the appended claims, the singular forms "a," "an," and "the" include plural referents and are used interchangeably with "at least one" and "one or more," unless the content clearly dictates otherwise.

[0112] The term "about," particularly in relation to a given amount, is meant to encompass deviations of plus or minus 5 percent.

[0113] The terms "comprises," "comprising," "includes," "including," "containing," "containing," and any variations thereof, as used herein, are intended to cover non-exclusive inclusions, and thus a process, method, product of a process, or composition that comprises, includes, or contains an element or list of elements, not only includes those elements, but can also include other elements that are not expressly listed or inherent to such process, method, product of a process, or composition.

[0114] The terms "bind," "binding," "interact," and "interacting," as used herein, refer to non-covalent interactions between macromolecules (e.g., between two polypeptides or between a polypeptide and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be "associated," "interacting," or "bound" (e.g., when molecule X is said to interact with molecule Y, it means that molecule X is non-covalently bound to molecule Y). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals, and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues of a DNA backbone), although some portions of a binding interaction may be sequence-specific.

[0115] The term "binding unit," as used herein, refers to one or more sites on a binding domain that non-covalently interact with a target peptide so that the target peptide associates with or binds to an engineered scaffold protein. In some cases, the portion of the target peptide that interacts with the binding unit is a disordered region. In some cases, the portion of the disordered region that interacts with the binding unit is a linear epitope. In some cases, when bound by an engineered scaffold protein, the bound portion is referred to as a "bound peptide."

[0116] The term "binding groove structure," as used herein, refers to the three-dimensional configuration of a protein domain characterized by a concave interface in a polypeptide lobe or a concave interface in at least two connected polypeptide lobes (wherein the concave interface lies between the two lobes), and in which binding of a target peptide induces a conformational change in which the lobe(s) at least partially fold together, burying the bound portion of the target peptide into the protein.

[0117] The term "clamshell structure," as used herein, refers to a three-dimensional structural feature of a binding monomer or multimer that includes two semi-symmetric helices supported by a beta sheet. A clamshell structure is an example of a three-dimensional structural feature having the binding groove structure described herein.

[0118] The term "disordered region," as used herein, refers to a segment of a protein that assumes a linear conformation at least for some time and / or under certain conditions. A linear segment of a protein may be part of a larger protein that may have a stable three-dimensional conformation under physiological conditions. An disordered region may also refer to a segment that lacks a distinctive three-dimensional structure at least for some time and / or under certain conditions. Some characteristics associated with intrinsically disordered regions (IDRs) include, but are not limited to, a higher percentage of polar or charged amino acids, a lower number of hydrophobic residues known to prevent cooperative folding, various three-dimensional states in dynamic equilibrium under physiological conditions, and the ability to exist in an ordered state or in association with binding partners, including proteins, nucleic acids, and lipids, under certain physiological conditions. See, e.g., M. Madan Babu, "The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease." Biochemical Society Transactions (2016). 44:1185-1200. For example, MTOR is a kinase with a predicted structure consisting of alpha helices, with some regions lacking predicted structure (i.e., disordered regions), and is an example of a protein with intrinsically disordered regions (IDRs). SDC-1 is another protein that is considered largely "unstructured" and has only short regions predicted to fold, and is also an example of an "intrinsically disordered protein" (IDP), in which most or all residues do not fold into a rigid structure.

[0119] The term "fused," as used herein, refers to at least two sequences that are joined together by a covalent bond (e.g., an amide or phosphodiester bond) or a linker, etc. The covalent bond can be formed by a conjugation (e.g., chemical or enzymatic) reaction.

[0120] The term "fusion partner," as used herein, refers to a molecule, agent, compound, macromolecule (e.g., a protein) that is fused to an engineered scaffold protein described herein. In some cases, the fusion partner can confer some function or activity to the fusion protein that is not provided by the engineered scaffold protein.

[0121] The term "fusion protein," as used herein, refers to a protein comprising at least two heterologous polypeptides. In some cases, the fusion protein comprises one or more engineered scaffold proteins and a fusion partner.

[0122] The term "hinge unit," as used herein, refers to a component of an engineered scaffold protein that stabilizes the structural conformation of the engineered scaffold protein described herein and / or stabilizes the binding interaction between a target peptide and an engineered scaffold protein described herein. The hinge unit can also at least partially bind to a target peptide described herein.

[0123] The term "immunoglobulin unit," as used herein, refers to an extracellular membrane-proximal peptide that is capable of recognizing an antigen, supporting a binding domain, and / or improving the structural integrity of the tertiary conformation of the engineered scaffold proteins described herein.

[0124] The term "linking unit," as used herein, refers to a flexible peptide that connects one or more components of a binding domain provided herein and allows for a structural conformational change in the engineered scaffold protein when the binding domain of the scaffold binds to a target peptide.

[0125] The term "target peptide," as used herein, refers to a peptide, a polypeptide, a protein domain, two or more protein domains, a peptide on the surface of a protein complex, or any combination thereof. A target peptide may be unmodified (post-translationally modified), endogenously or exogenously modified.

[0126] The term "wild-type counterpart" or "WT counterpart," as used herein, refers to a protein or portion of a protein from which an engineered protein is derived. For example, a WT counterpart of an engineered scaffold protein described herein can be the WT protein from which the engineered scaffold protein is derived, prior to modification for use in accordance with the present disclosure. In another example, a WT counterpart of a binding domain described herein can be a domain or region of the WT protein from which the binding domain is derived, prior to modification for use in accordance with the present invention, which WT protein may be separate and / or different from the WT counterpart of an engineered scaffold protein described herein.

[0127] Engineered scaffold proteins Compositions and methods comprising engineered scaffold proteins or their use are provided herein. The engineered scaffold proteins provided herein can bind to a known sequence of amino acids. In some embodiments, the known sequence of amino acids can be a disordered region in a target peptide described herein. Such target peptides can have one or more disordered regions described herein in combination with one or more ordered regions. Binding can be precise, specific, sequence-specific, or any combination thereof. Binding of a target peptide to an engineered scaffold protein described herein can induce one or more activities in vivo and / or in vitro. For example, engineered scaffold proteins and compositions comprising the same can locally and sequence-specifically edit protein structure or function, modulate protein-polymer interaction networks, sense and detect biological analytes, induce local conformational changes, specifically bind to two or more targets simultaneously by fusing and linking multiple binding moieties together, bind to macromolecular targets in the extracellular matrix and intracellular environment, sense and detect biological molecules, and the like.

[0128] In some embodiments, the engineered scaffold proteins provided herein can be derived from the scaffold proteins described herein. The scaffold proteins disclosed herein demonstrate binding affinity for specific peptide sequences that may be contained in disordered regions and can be used as a starting point for designing the engineered scaffold proteins disclosed herein based on the requirements of a therapeutic modality, such as the size of the binder, the sequence to be bound, and physiochemical properties such as solubility and temperature sensitivity. Thus, "scaffold protein" refers to a protein used as a starting point for generating an "engineered scaffold protein." Such engineered scaffold proteins have modified binding properties, such as specificity for the disordered regions of a target protein, relative to the starting scaffold protein. For example, a scaffold protein may be identified from any of the proteins listed in Table 1 based on the peptide sequence to which the scaffold protein is known to bind, such as those listed in Table 3, and the disordered region of the target protein, and may be modified or engineered to achieve specific binding of the disordered region (e.g., high affinity binding), a desired size of the engineered scaffold protein or its fusion protein, or a desired specific physiochemical property (e.g., solubility and temperature) of the engineered scaffold protein or its fusion protein. For further examples of proteins known to form complexes with peptide sequences and protein classes that can serve as starting points for the scaffolds disclosed herein, see, e.g., Martins, PM, Santos, LH, Mariano, D. et al. Propedia: a database for protein-peptide identification based on a hybrid clustering algorithm. BMC Bioinformatics 22, 1 (2021).

[0129] In some embodiments, the engineered scaffold proteins provided herein comprise an amino acid sequence of less than about 100 amino acids, between about 100 amino acids and about 500 amino acids, or more than about 500 amino acids. In some embodiments, the engineered scaffold proteins provided herein comprise an amino acid sequence of at least about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 350 amino acids, about 400 amino acids, about 450 amino acids, about 500 amino acids, or more than 500 amino acids. In some embodiments, the engineered scaffold proteins provided herein comprise an amino acid sequence of at least about 160 amino acids, about 180 amino acids, about 200 amino acids, about 220 amino acids, about 240 amino acids, about 260 amino acids, about 280 amino acids, about 300 amino acids, about 320 amino acids, about 340 amino acids, about 360 amino acids, about 380 amino acids, about 400 amino acids, about 420 amino acids, or about 440 amino acids. In certain embodiments, the engineered scaffold proteins described herein are of a size suitable for packaging into a vector, such as a viral vector. For example, in some embodiments, the engineered scaffold proteins described herein are less than 250 amino acids.

[0130] In some embodiments, the engineered scaffold proteins provided herein have a protein isoelectric (pi) point of less than about 4 pi, between about 4 pi and about 8 pi, or greater than 8 pi. In some embodiments, the engineered scaffold proteins provided herein have a pi point of about 4 pi, about 4.2 pi, about 4.4 pi, about 4.6 pi, about 4.8 pi, about 5 pi, about 5.2 pi, about 5.4 pi, about 5.6 pi, about 5.8 pi, about 6 pi, about 6.2 pi, about 6.4 pi, about 6.6 pi, about 6.8 pi, about 7 pi, about 7.2 pi, about 7.4 pi, about 7.6 pi, about 7.8 pi, or about 8 pi. Those of skill in the art will know how to determine the pi points of the engineered scaffold proteins described herein.

[0131] The binding of engineered scaffold proteins or their therapeutic molecules can be characterized by describing their affinity to target peptides.In some embodiments, the affinity of the engineered scaffold proteins or their fusion proteins described herein to their target proteins is less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, less than 100 fM, or less than 1 aM.Such affinity can be achieved by engineered scaffold proteins or their fusion proteins that are specific to a particular disordered region of the target protein.

[0132] In some embodiments, the engineered scaffold proteins provided herein comprise one or more amino acid changes compared to their WT counterparts. In some embodiments, the one or more amino acid changes comprise one amino acid change, two amino acid changes, three amino acid changes, four amino acid changes, five amino acid changes, six amino acid changes, seven amino acid changes, eight amino acid changes, nine amino acid changes, ten amino acid changes, eleven amino acid changes, twelve amino acid changes, thirteen amino acid changes, fourteen amino acid changes, fifteen amino acid changes, sixteen amino acid changes, seventeen amino acid changes, eighteen amino acid changes, nineteen amino acid changes, twenty amino acid changes, or more amino acid changes compared to their WT counterparts.

[0133] In some embodiments, the one or more amino acid changes are one or more amino acid substitutions, deletions, insertions, or any combination thereof. In some embodiments, the one or more amino acid substitutions are one or more conservative substitutions, one or more non-conservative substitutions, or a combination thereof. In some embodiments, the one or more amino acid changes are one or more conservative substitutions. A conservative substitution as described herein refers to the substitution of one amino acid for another amino acid, such as within a family of amino acids whose side chains are related. Conversely, the term "non-conservative substitution," as used herein, refers to the substitution of one amino acid residue for another amino acid residue that does not have a related side chain. Genetically encoded amino acids can be divided into four families with related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) nonpolar (hydrophobic): Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W) ), Gly(G), Tyr(Y), where non-polar is also further divided: (i) strongly hydrophobic: Ala(A), Val(V), Leu(L), Ile(I), Met(M), Phe(F), and (ii) moderately hydrophobic: Gly(G), Pro(P), Cys(C), Tyr(Y), Trp(W), and (4) uncharged polar: Asn(N), Gln(Q), Ser(S), Thr(T). Amino acids may be associated with aliphatic side chains: Gly(G), Ala(A), Val(V), Leu(L), Ile(I), Ser(S), Thr(T), where Ser(S) and Thr(T) are optionally classified separately as aliphatic hydroxyls. The amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). The amino acids may be related by amide side chains: Asn (N), Gln (Q). The amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M). In some embodiments, the one or more amino acid changes are one or more non-conservative substitutions.In some embodiments, the one or more amino acid changes are substitutions of one or more hydrophobic residues with one or more charged or polar residues, or a combination thereof. For example, one or more of Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), or Tyr (Y) may be substituted with one or more of Asp (D), Glu (E), Lys (K), Arg (R), His (H), Asn (N), Gln (Q), Ser (S), Thr (T), or any combination thereof.

[0134] In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in increased binding activity, including more selective binding, more frequent binding, more rapid binding, longer duration of binding, binding with greater affinity, or binding with any combination of the foregoing, compared to the corresponding WT. In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in reduced immunogenicity when administered to a subject, compared to the corresponding WT. In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in increased solubility, compared to the corresponding WT. In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in increased stability, compared to the corresponding WT. In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in altered hydrophobicity, compared to the corresponding WT. The altered hydrophobicity, compared to the corresponding WT, can be increased or decreased hydrophobicity, depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in altered hydrophilicity, compared to the corresponding WT. The altered hydrophilicity compared to the corresponding WT can be increased or decreased hydrophilicity depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid changes in the engineered scaffold proteins provided herein can result in altered surface charge compared to the corresponding WT. The altered surface charge compared to the corresponding WT can be increased or decreased surface charge depending on the desired activity of the engineered scaffold protein. The desired activity can be related to binding activity, solubility, etc. as described herein.

[0135] In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 60% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 65% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 70% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 75% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 80% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 85% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 90% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 95% identical to any one of the amino acid sequences set forth in Table 6. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is 100% identical to any one of the amino acid sequences set forth in Table 6.

[0136] In some embodiments, the engineered scaffold proteins provided herein comprise one or more amino acid changes compared to any one of the amino acid sequences set forth in Table 6. In some embodiments, the one or more amino acid changes comprise 1 amino acid change, 2 amino acid changes, 3 amino acid changes, 4 amino acid changes, 5 amino acid changes, 6 amino acid changes, 7 amino acid changes, 8 amino acid changes, 9 amino acid changes, 10 amino acid changes, 11 amino acid changes, 12 amino acid changes, 13 amino acid changes, 14 amino acid changes, 15 amino acid changes, 16 amino acid changes, 17 amino acid changes, 18 amino acid changes, 19 amino acid changes, 20 amino acid changes, or more amino acid changes compared to any one of the amino acid sequences set forth in Table 6. In some embodiments, the engineered scaffold proteins comprising one or more amino acid changes are variants of the engineered scaffold proteins described herein. It is understood that reference to an engineered scaffold protein also refers to the engineered scaffold protein variants described herein.

[0137] In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in increased binding activity, including more selective binding, more frequent binding, more rapid binding, longer duration of binding, binding with greater affinity, or any combination of the foregoing, as compared to any one of the amino acid sequences set forth in Table 1. In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in decreased immunogenicity when administered to a subject as compared to any one of the amino acid sequences set forth in Table 1. In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in increased solubility as compared to any one of the amino acid sequences set forth in Table 1. In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in increased stability as compared to any one of the amino acid sequences set forth in Table 1. In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in an altered hydrophobicity as compared to any one of the amino acid sequences set forth in Table 1. The altered hydrophobicity can be an increase in hydrophobicity or a decrease in hydrophobicity as compared to any one of the amino acid sequences set forth in Table 1, depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in an alteration in hydrophilicity compared to any one of the amino acid sequences set forth in Table 1. The alteration in hydrophilicity can be an increase in hydrophilicity or a decrease in hydrophilicity compared to any one of the amino acid sequences set forth in Table 1, depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid changes in an engineered scaffold protein provided herein can result in an alteration in surface charge compared to any one of the amino acid sequences set forth in Table 1. The alteration in surface charge can be an increase in surface charge or a decrease in surface charge compared to any one of the amino acid sequences set forth in Table 1, depending on the desired activity of the engineered scaffold protein.

[0138] In some embodiments, the compositions and methods described herein involve the use of one or more engineered scaffold proteins. In such embodiments, the engineered scaffold proteins may interact with or bind to one target peptide or two or more target peptides. In some embodiments, the compositions and methods described herein involve the use of two, three, or four or more engineered scaffold proteins, where the engineered scaffold proteins target one, two, three, four, or more target peptides.

[0139] In some embodiments, the engineered scaffold proteins provided herein comprise one or more of a binding domain, an immunoglobulin unit, a linker, a fusion partner, an epitope masking unit, or any combination thereof.

[0140] In some embodiments, the engineered scaffold proteins provided herein are also engineered to reduce the immunogenicity of the engineered scaffold proteins. For example, in some embodiments, the scaffold proteins described herein are selected as the scaffold proteins from which the engineered scaffold proteins are derived by determining common genes, such as common HLA alleles. In another example, the engineered scaffold protein includes one or more amino acid changes. In some embodiments, such changes are amino acid changes that can be buried or distal to the surface of the binding domain, so that the binding unit can be effective and selective for the target peptides described herein. In yet another example, the engineered scaffold protein can include or use an epitope masking unit to hide one or more amino acid changes. In a further example, the engineered scaffold protein may not include one or more immunoglobulin units, which can function to prevent direct binding and induce an immune response.

[0141] Binding domain Provided herein are compositions and methods comprising engineered scaffold proteins or uses thereof, wherein the engineered scaffold proteins comprise one or more binding domains. In some embodiments, the binding domains described herein can be engineered to bind to the target peptides described herein. In some embodiments, the binding domains described herein can be engineered to bind to disordered regions in the target peptides described herein.

[0142] In some embodiments, the engineered scaffold proteins described herein comprise one, two, three, four, or more binding domains. In some embodiments, the engineered scaffold proteins comprise two or more binding domains, such binding domains may be covalently attached, attached by one or more linkers, or a combination thereof. Examples of the organization of binding domains in the constructs described herein are described in Example 4.

[0143] In some embodiments, a binding domain comprises one or more binding units, hinge units, linking units, linkers, or combinations thereof.

[0144] Combined Unit In some embodiments, the engineered scaffold proteins provided herein comprise a binding domain comprising one or more binding units. In some embodiments, the binding units bind to the target peptide non-covalently. In some embodiments, the binding units bind to one or more target peptides non-covalently. In some embodiments, the binding units bind to the disordered region of the target peptide. In some embodiments, the binding units bind to the target peptide in a sequence-specific manner.

[0145] In some embodiments, the binding units provided herein comprise one or more amino acids capable of binding to one or more amino acids of a target peptide. In some embodiments, the binding units described herein comprise one or more amino acids capable of binding to one or more amino acids in an irregular region of a target peptide. In some embodiments, the binding units described herein comprise one or more amino acids capable of sequence-specifically binding to one or more amino acids in an irregular region of a target peptide. It is understood that not all of the one or more amino acid residues capable of binding to a target peptide must be bound by one or more amino acids in an irregular region of a target peptide to produce sequence specificity. In some embodiments, one or more amino acid residues capable of binding to a target peptide may not be contiguous in the primary structure of the engineered scaffold protein described herein, but may be included in the binding unit of the engineered scaffold protein when the engineered scaffold protein assumes a tertiary structure. In some embodiments, one or more amino acids in the binding unit capable of binding to a target peptide are oriented in the binding unit to optimize binding ability to the irregular region of the target peptide. In some embodiments, one or more amino acids of the binding unit that can bind to target peptide are antisense to one or more amino acids of the disordered region in the target peptide, as determined by corresponding sense-antisense amino acid pairing.Such amino acid pairing is described in Stambuk et al., Theory in Biosciences, 123(4):265-275(2005).In some embodiments, one or more amino acids of the binding unit that can bind to target peptide form at least one non-covalent interaction with one or more amino acids of the disordered region in the target peptide, as determined by corresponding sense-antisense amino acid pairing.

[0146] In some embodiments, when a binding unit folds into a tertiary structure, the binding unit can have a three-dimensional conformation comprising one or more amino acids that are antisense to one or more amino acids in the disordered region of the target peptide, as determined by corresponding sense-antisense amino acid pairing. In some embodiments, when a binding unit folds into a tertiary structure, the binding unit can have a three-dimensional conformation comprising one or more amino acids that can form at least one non-covalent interaction with one or more amino acids in the disordered region of the target peptide, as determined by corresponding sense-antisense amino acid pairing.

[0147] In some embodiments, the binding units provided herein have an extended conformation. In some embodiments, the binding units provided herein comprise one or more secondary structure motifs. In some embodiments, the binding units comprise one or more helices. Suitable helices include an alpha helix, a 3.10 helix, and a pi helix. In some embodiments, the binding units provided herein comprise one or more helices. In some embodiments, the binding units provided herein comprise one or more, two or more, three or more, four or more, or five or more helices. In some embodiments, the binding domains described herein comprise two binding units, each binding unit being a helix. In some embodiments, the binding domains described herein comprise two helices. In some embodiments, the binding domains described herein comprise two helices, and the two helices are semi-symmetric. In some embodiments, where the binding domain comprises two helices, the helices run antiparallel in the three-dimensional conformation of the binding domain.

[0148] In some embodiments, the binding units provided herein comprise one or more alpha helices. In some embodiments, the binding units provided herein comprise one or more, two or more, three or more, four or more, or five or more alpha helices. In some embodiments, the binding domains described herein comprise two binding units, each binding unit being an alpha helix. In some embodiments, the binding domains described herein comprise two alpha helices. In some embodiments, when the binding domain comprises two alpha helices, the alpha helices run antiparallel in the three-dimensional structure of the binding domain.

[0149] In some embodiments, a binding unit comprises an amino acid sequence of less than 40 amino acids, 40 to 200 amino acids, or more than 200 amino acids. In some embodiments, a binding unit provided herein comprises an amino acid sequence of about 40 amino acids, 60 amino acids, 80 amino acids, 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, or 200 amino acids in length. In some embodiments, a binding unit provided herein comprises an amino acid sequence of about 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 105 amino acids, or about 110 amino acids.

[0150] In some embodiments, the binding unit provided herein comprises one or more amino acid changes compared to its WT counterpart.In some embodiments, the binding unit comprising one or more amino acid changes is a variant of the binding unit described herein.It is understood that reference to a binding unit also refers to the binding unit variant described herein.In some embodiments, the one or more amino acid changes comprise one amino acid change, two amino acid changes, three amino acid changes, four amino acid changes, five amino acid changes, six amino acid changes, seven amino acid changes, eight amino acid changes, nine amino acid changes, ten amino acid changes, eleven amino acid changes, twelve amino acid changes, thirteen amino acid changes, fourteen amino acid changes, fifteen amino acid changes, sixteen amino acid changes, seventeen amino acid changes, eighteen amino acid changes, nineteen amino acid changes, twenty amino acid changes or more amino acid changes compared to its WT counterpart. In some embodiments, one or more amino acid changes in a binding unit provided herein can result in increased binding activity, including more selective binding, more frequent binding, more rapid binding, longer duration of binding, binding with greater affinity, or binding with some combination of the foregoing, compared to the WT counterpart.

[0151] In some embodiments, a binding unit comprises an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any one of the sequences set forth in Table 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 60% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 65% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 70% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 75% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 80% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, the binding units provided herein comprise an amino acid sequence that is at least about 85% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, the binding units provided herein comprise an amino acid sequence that is at least about 90% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, the binding units provided herein comprise an amino acid sequence that is at least about 95% identical to any one of the amino acid sequences set forth in Table 4. In some embodiments, the binding units provided herein comprise an amino acid sequence that is 100% identical to any one of the amino acid sequences set forth in Table 4.

[0152] In some embodiments, the binding units provided herein comprise one or more amino acid changes compared to any one of the amino acid sequences set forth in Table 4. In some embodiments, the one or more amino acid changes comprise 1 amino acid change, 2 amino acid changes, 3 amino acid changes, 4 amino acid changes, 5 amino acid changes, 6 amino acid changes, 7 amino acid changes, 8 amino acid changes, 9 amino acid changes, 10 amino acid changes, 11 amino acid changes, 12 amino acid changes, 13 amino acid changes, 14 amino acid changes, 15 amino acid changes, 16 amino acid changes, 17 amino acid changes, 18 amino acid changes, 19 amino acid changes, 20 amino acid changes, or more amino acid changes compared to any one of the amino acid sequences set forth in Table 4. In some embodiments, the one or more amino acid changes in the binding units provided herein can result in increased binding activity, including more selective binding, more frequent binding, more rapid binding, longer duration of binding, binding with greater affinity, or binding with some combination of the foregoing, compared to any one of the amino acid sequences set forth in Table 4.

[0153] Hinge Unit In some embodiments, the engineered scaffold proteins provided herein comprise a binding domain comprising one or more hinge units. In some embodiments, the hinge unit stabilizes the structural conformation of the engineered scaffold protein in an open, unbound state, the engineered scaffold protein in an open, unbound state, the engineered scaffold protein in a closed, unbound state, the engineered scaffold protein in a closed, bound state, the engineered scaffold protein in a partially bound state, or any combination thereof. The hinge unit can also bind to one or more target peptides. In some embodiments, the hinge unit can also be engineered to bind non-covalently to one or more target peptides. In some embodiments, the hinge unit can bind to an irregular region in the target peptide. In some embodiments, the hinge unit can bind to the target peptide in a sequence-specific manner. In some embodiments, the hinge unit and the binding unit can function together cooperatively, cooperatively, and / or synergistically to bind to the target peptide.

[0154] In some embodiments, the binding domains provided herein comprise one or more hinge units. In some embodiments, the binding domains provided herein comprise one, two, three, four, five, or more hinge units described herein. In some embodiments, the hinge units have a linear or partially linear three-dimensional conformation. In some embodiments, the hinge units have a concave or partially concave three-dimensional conformation. In some embodiments, the hinge units have a convex or partially convex three-dimensional conformation. In some embodiments, the hinge units have a planar or partially planar three-dimensional conformation. In some embodiments, the hinge units described herein have a conformation that is complementary or partially complementary to the irregular region in the target peptide as determined by corresponding sense-antisense amino acid pairing. In some embodiments, the hinge units described herein have a flexible or partially flexible structure that matches the three-dimensional structure of the irregular region in the target peptide described herein. In some embodiments, the hinge units have non-covalent surface forces or charges that can facilitate binding of the target peptide to the binding domain. Non-covalent surface forces or charges include the hydrophobicity, hydrophilicity, and polarity of the surface of the hinge unit.

[0155] In some embodiments, the hinge unit comprises one or more linear peptide chains, secondary structural motifs (e.g., beta sheets), one or more covalent interactions (e.g., disulfide bonds), one or more non-covalent interactions (e.g., hydrogen bonds, salt bridges, etc.), chemical agents (e.g., DMSO, PEG, starch, etc.), or combinations thereof.

[0156] In some embodiments, one or more hinge units are connected to one or more linkage units, hi some embodiments, the linkage domain comprises two linkage units and one hinge unit, and the two linkage units are connected by the hinge unit.

[0157] In some embodiments, the hinge units provided herein comprise one or more secondary structure motifs. In some embodiments, the hinge units provided herein comprise one or more β-sheet proteins. In some embodiments, the one or more β-sheet proteins form one or more β-sheets. In some embodiments, the one or more β-sheets each comprise from about 2 to about 20 or more β-sheet protein strands. In some embodiments, the one or more β-sheets each comprise from about 4, 5, 6, 7, 8, 9, 10, 11, or about 12 β-sheet protein strands. In some embodiments, the one or more β-sheets each comprise from about 6 to about 10 β-sheet protein strands. In some embodiments, the hinge units provided herein comprise one or more, two or more, three or more, four or more, or five or more β-sheets.

[0158] In some embodiments, the hinge unit comprises an amino acid sequence of less than 40 amino acids, 40 to 200 amino acids, or more than 200 amino acids. In some embodiments, the hinge units provided herein comprise an amino acid sequence of about 40 amino acids, 60 amino acids, 80 amino acids, 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, or 200 amino acids in length. In some embodiments, the hinge units provided herein comprise an amino acid sequence of about 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 105 amino acids, or about 110 amino acids.

[0159] In some embodiments, the hinge unit provided herein comprises one or more amino acid changes compared to its WT counterpart.In some embodiments, the hinge unit comprising one or more amino acid changes is a variant of the hinge unit described herein.It is understood that reference to a hinge unit also refers to the hinge unit variant described herein.In some embodiments, the one or more amino acid changes comprise one amino acid change, two amino acid changes, three amino acid changes, four amino acid changes, five amino acid changes, six amino acid changes, seven amino acid changes, eight amino acid changes, nine amino acid changes, ten amino acid changes, eleven amino acid changes, twelve amino acid changes, thirteen amino acid changes, fourteen amino acid changes, fifteen amino acid changes, sixteen amino acid changes, seventeen amino acid changes, eighteen amino acid changes, nineteen amino acid changes, twenty amino acid changes or more amino acid changes compared to its WT counterpart. In some embodiments, one or more amino acid changes in the hinge unit provided herein can result in increased binding activity, including more selective binding, more frequent binding, more rapid binding, longer duration of binding, binding with greater affinity, or binding with some combination of the foregoing, compared to the WT counterpart.

[0160] In some embodiments, the hinge unit comprises an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any one of the sequences set forth in Table 5. In some embodiments, the hinge unit provided herein comprises an amino acid sequence that is at least about 60% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge unit provided herein comprises an amino acid sequence that is at least about 65% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge unit provided herein comprises an amino acid sequence that is at least about 70% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge unit provided herein comprises an amino acid sequence that is at least about 75% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge unit provided herein comprises an amino acid sequence that is at least about 80% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge units provided herein comprise an amino acid sequence that is at least about 85% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge units provided herein comprise an amino acid sequence that is at least about 90% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge units provided herein comprise an amino acid sequence that is at least about 95% identical to any one of the amino acid sequences set forth in Table 5. In some embodiments, the hinge units provided herein comprise an amino acid sequence that is 100% identical to any one of the amino acid sequences set forth in Table 5.

[0161] In some embodiments, the hinge units provided herein comprise one or more amino acid changes compared to any one of the amino acid sequences set forth in Table 5. In some embodiments, the one or more amino acid changes comprise 1 amino acid change, 2 amino acid changes, 3 amino acid changes, 4 amino acid changes, 5 amino acid changes, 6 amino acid changes, 7 amino acid changes, 8 amino acid changes, 9 amino acid changes, 10 amino acid changes, 11 amino acid changes, 12 amino acid changes, 13 amino acid changes, 14 amino acid changes, 15 amino acid changes, 16 amino acid changes, 17 amino acid changes, 18 amino acid changes, 19 amino acid changes, 20 amino acid changes, or more amino acid changes compared to any one of the amino acid sequences set forth in Table 5. In some embodiments, the one or more amino acid changes in the hinge units provided herein can result in increased binding activity, including more selective binding, more frequent binding, more rapid binding, longer binding, binding with greater affinity, or binding with any combination of the foregoing, compared to any one of the amino acid sequences set forth in Table 5.

[0162] In some embodiments, the one or more linking units and the one or more hinge units may be covalently connected, connected by a linking unit, or connected by a linker as described herein. In some embodiments, the one or more linking units and the one or more hinge units are monomers. In some embodiments, the hinge unit is connected or attached to the N-terminus of the linking unit. In some embodiments, the one or more linking units and the one or more hinge units are connected as monomers. In some embodiments, the one or more linking units and the one or more hinge units are multimers. In some embodiments, the one or more linking units and the one or more hinge units are multimers, and in the presence of a target peptide, the one or more linking units and the one or more hinge units form a binding domain.

[0163] Connecting unit In some embodiments, the engineered scaffold proteins provided herein comprise a binding domain comprising one or more linking units. In some embodiments, the linking units comprise one or more of a beta-sheet strand, an alpha helix, a linear peptide, or a combination thereof. In some embodiments, the linking units described herein connect a set of binding domain components. For example, one or more linking units each connect a set of binding domain components comprising a linking unit and a hinge unit, and one or more linking units are located between the sets. In some embodiments, the engineered scaffolds provided herein comprise one, two, three, four, five, or more linking units. Linking units, as described in detail herein, may also be referred to as linkers.

[0164] Immunoglobulin Unit In some embodiments, the engineered scaffold proteins provided herein comprise a binding domain comprising one or more immunoglobulin units. In some embodiments, the engineered scaffolds comprising the immunoglobulin units described herein may be useful in compositions or methods for inducing an immune response. In some embodiments, the engineered scaffolds provided herein comprise one or two immunoglobulin units. The immunoglobulin units described herein can be connected to one or more of the binding units and / or hinge units covalently, by one or more linking units, or by one or more linkers described herein.

[0165] In some embodiments, the immunogenicity of the engineered scaffold proteins described herein is improved by removing one or more immunoglobulin units, thereby reducing the risk of an autoimmune response.

[0166] Binding groove structure In some embodiments, the binding domain has a three-dimensional structure referred to herein as a binding groove structure. In some embodiments, the binding groove structure comprises one or more polypeptide lobes and a binding groove. In some embodiments, each of the polypeptide lobes of the binding groove structure comprises one or more binding units provided herein. In some embodiments, the binding groove of the binding groove structure comprises one or more hinge units described herein. In some embodiments, the lobes, binding grooves, or components thereof are connected by one or more linkage units and / or linkers described herein. Thus, in some embodiments, the engineered scaffolds having a binding groove structure provided herein comprise one or more of a binding unit, a hinge unit, a linkage unit, a linker, or any combination thereof. Three-dimensional renderings of binding groove structures can be seen in Figures 33 and 34.

[0167] Scaffold proteins The engineered scaffold protein provided herein or its components can be derived from a scaffold protein.In some embodiments, each component of the engineered scaffold protein provided herein is independently derived from a scaffold protein.Therefore, each of the binding domain, binding unit, hinge unit, linking unit, immunoglobulin unit, epitope masking unit, etc. is derived from a scaffold protein or its components.Such scaffold protein or its components can serve as the wild-type counterpart or experimental control of the corresponding component when comparable evaluation indexes are described herein.

[0168] In some embodiments, the engineered scaffold proteins or components thereof provided herein are derived from a scaffold protein listed in Table 1. In some embodiments, the engineered scaffold protein is an engineered MHC II monomer capable of binding to a disordered region in a complete protein. In some embodiments, the engineered scaffold protein is an engineered MHC I molecule capable of binding to a disordered region in a complete protein. In some embodiments, the engineered scaffold protein is an MHC-like protein engineered to bind to a disordered region in a complete protein. In some embodiments, the engineered scaffold protein is an engineered zinc finger binding protein capable of binding to a linear epitope in a complete protein. In some embodiments, the engineered scaffold protein is an engineered caspase protein capable of binding to a linear epitope in a complete protein. In some embodiments, the engineered scaffold protein is an engineered protein ligase, including biotin ligase, sortase, subtilisin-derived enzymes, etc. In some embodiments, the engineered scaffold protein is an engineered protease derived from TEV, enterokinase, thrombin, factor Xa, etc. In some embodiments, the engineered scaffold protein is an engineered ACT domain protein. In some embodiments, the engineered scaffold protein is an engineered kinase and / or phosphatase. In some embodiments, the engineered scaffold protein is an engineered chaperone protein.

[0169] Creation and Design In some embodiments, engineered scaffold proteins are designed to bind to the target peptides described herein.As described herein, scaffold proteins can be selected as the binding proteins from which engineered scaffold proteins are derived.The selection of scaffolds can be based on the type of molecule to be bound (e.g., DNA, RNA, or protein), known interactions with the molecule (e.g., binding selectivity, binding strength, binding affinity, etc.), the immunogenicity of the scaffold, the function or biological activity of the scaffold, etc.After selecting the scaffold protein, the amino acid sequence of the engineered scaffold protein can be generated to bind to the target peptide.

[0170] Therefore, in some embodiments, also provided herein is a method for generating the amino acid sequence of engineered scaffold protein that binds to the disordered region of target peptide.In some embodiments, the method comprises selecting one or more scaffold protein sequences.Selection of one or more scaffold protein sequences can be carried out by any suitable technique, for example, by mining from metagenomic database.In some embodiments, to select two or more scaffold proteins, sequence homology search can be carried out (for example, protein domain search, pairwise alignment or HMM-based alignment).

[0171] In some embodiments, the methods include evaluating one or more scaffold protein sequences for a desired function, activity, and / or characteristic. In some embodiments, evaluating one or more scaffold protein sequences for a desired function, activity, and / or characteristic includes weighting factors related to ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or a combination thereof, of the scaffold protein(s) to generate amino acid sequences of engineered scaffold proteins that are predicted to have one or more enhanced or improved desired characteristics, including ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or a combination thereof. Examples of such weighted factors include the three-dimensional conformation of the scaffold protein(s), individual protein domains, amino acid sequence, charge, polarity, hydrophobicity / hydrophilicity, and / or acidity / basicity of certain amino acid residues (e.g., amino acid residues that interact with or bind to a ligand), etc. In some embodiments, weighting one or more factors of the scaffold protein comprises assigning a value to one or more weighted factors based on an estimated probability of improving one or more desired characteristics, assigning a value to one or more weighted factors and measuring deviation of the value relative to a target value or threshold or a value assigned to such factor of a different scaffold protein, or both. In some embodiments, the method further comprises predicting whether the generated amino acid sequence represents an engineered scaffold protein exhibiting improved characteristics, whether one or more changes in one or more weighted factors improve the value relative to a target value or threshold or a value assigned to such factor of a different scaffold protein, or both. The different scaffold protein can be a naturally occurring scaffold protein, a WT counterpart, or a second scaffold protein in cases where more than one scaffold protein is selected in the methods described herein.In some embodiments, the one or more changes comprise one or more amino acid changes.

[0172] In some embodiments, the methods include selecting and evaluating one or more scaffold protein sequences described herein and engineering the scaffold protein sequence to bind to a linear epitope in a disordered region of a target peptide. In certain embodiments, the engineered scaffold protein sequence, such as the binding unit, or in some embodiments, the hinge unit, or both, of the engineered scaffold protein, is also engineered based on an estimated probability of having improved one or more desired characteristics (e.g., improved ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or a combination thereof). Engineering the sequence of the engineered scaffold protein can be performed by suitable methods, such as computational, rational, or directed evolution methods. Examples include site-directed mutagenesis, rational design, domain swapping, and ancestral sequence reconstruction of the corresponding scaffold protein(s). Thus, the methods described herein generate engineered scaffold proteins that contain one or more alterations or one or more amino acid changes compared to their WT counterparts and / or corresponding scaffold proteins.

[0173] In some embodiments, a method comprises manipulating and / or generating amino acid sequence(s) of an engineered scaffold protein(s) by performing a method described herein, in some embodiments, the method further comprises manipulating and / or generating amino acid sequences based on the selected scaffold protein(s), wherein the generated amino acid sequences represent an engineered scaffold protein predicted to have enhanced or improved ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or a combination thereof.

[0174] In some embodiments, evaluating one or more scaffold protein sequences comprises manually evaluating the amino acid sequences of one or more scaffold protein sequences. In some embodiments, the method comprises evaluating one or more scaffold protein sequences in a machine learning model. In some embodiments, the machine learning model used herein is trained to enhance one or more desired features of the scaffold protein. In some embodiments, the machine learning model used herein is a generative model, a deep learning model, or the like.

[0175] In some embodiments, the method further comprises a second iteration of evaluating one or more desired characteristics of the generated engineered scaffold protein (e.g., ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or a combination thereof), weighting one or more weighted factors of the generated engineered scaffold protein (e.g., three-dimensional conformation, individual protein domains, charge, polarity, hydrophobicity / hydrophilicity, and / or acidity / basicity of certain amino acid residues (e.g., amino acid residues that interact with or bind to a ligand), etc., or a combination thereof), or both, and providing information for further generation of engineered scaffold proteins. In some embodiments, the above-described method steps may be repeated two or more times or multiple times.

[0176] In some embodiments, the method further comprises assaying the generated engineered scaffold protein. In some embodiments, the method further comprises assaying the generated engineered scaffold protein in silico and simulating data regarding ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or combinations thereof, and weighting the evaluation index relative to the pre-predicted results. In some embodiments, the method further comprises assaying the generated engineered scaffold protein in an in vitro or in vivo assay and obtaining data regarding ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or combinations thereof, and weighting the data relative to the pre-predicted results. In some embodiments, the method further comprises evaluating the generated engineered scaffold protein in a machine learning model and predicting an outcome with respect to ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, etc., or a combination thereof, and weighting the data relative to the prior predicted outcome.

[0177] Thus, in some embodiments, methods for obtaining an engineered scaffold protein or a fusion protein thereof that binds to a target peptide are described herein. In some embodiments, the method comprises contacting a target peptide with an engineered scaffold protein, a fusion protein thereof, a nucleic acid encoding it, a host cell expressing it, or a library comprising any of the foregoing, under conditions that allow for the formation of an engineered scaffold protein:target peptide complex. In some embodiments, the method further comprises obtaining, from the engineered scaffold protein:target peptide complex, an engineered scaffold protein scaffold that binds to the target peptide, and preferably an engineered scaffold protein scaffold that has improved binding activity for the target peptide compared to its scaffold protein counterpart.

[0178] In some embodiments, the method comprises obtaining at least two engineered scaffold proteins that bind to one or more target peptides. In some embodiments, the method comprises contacting one or more target peptides with an engineered scaffold protein, a fusion protein thereof, a nucleic acid encoding it, a host cell expressing it, or a library comprising any of the foregoing, under conditions that allow for the formation of an engineered scaffold protein:target peptide complex. In some embodiments, the method further comprises combining the engineered scaffold protein:target peptide complex with a cross-linking agent, where cross-linking of the engineered scaffold protein:target peptide complex elicits a detectable response. Examples of cross-linking agents include antibodies, antibody fragments, binding peptides, or epitope tags. In some embodiments, the method further comprises obtaining the engineered scaffold proteins that bind to one or more target peptides from the complex. In some embodiments, two or more engineered scaffold proteins bind to the same epitope of the target peptide or distinct epitopes of the target peptide. In some embodiments, the evaluating and / or assaying methods described herein further comprise evaluating the engineered scaffold protein or analyzing the engineered scaffold protein fused to a fusion partner and / or heterologous agent. Fusion partners and heterologous agents are further described herein.

[0179] In some embodiments, results from in silico, in vitro, in vivo assays, and / or machine learning models can be used herein to inform further or other evaluation of the scaffold protein(s) and / or generation of subsequent iterations of engineered scaffold proteins. In other embodiments, where the methods involve the use of machine learning models, the methods include utilizing results from the in silico, in vitro, in vivo assays, and / or machine learning models described herein to retrain the machine learning model for further evaluation of the scaffold protein. In some embodiments, the machine learning model can be retrained for multiple iterations. In some embodiments, retraining the machine learning model includes minimizing and / or optimizing one or more functions, weights, parameters, variables, features, etc. of the machine learning model. In some embodiments, methods of generating amino acid sequences for engineered scaffold proteins using the retrained machine learning model are also described herein.

[0180] In some embodiments, the method can be implemented in a computer-readable medium. Accordingly, in some embodiments, a system for implementing a method for generating amino acid sequences of engineered scaffold proteins that bind to disordered regions of a target peptide is also described herein. Such a system can include a computer-readable medium comprising a computer-readable memory capable of storing instructions for implementing the methods described herein. The computer-readable memory can be local (e.g., a hard drive) or online (e.g., the cloud). For example, the computer-readable memory can include instructions for evaluating scaffold protein(s), instructions for generating amino acid sequences of engineered scaffold protein(s), or both. In some embodiments, the system described herein is a computer system that utilizes the computer-readable medium described herein and further includes a processor operably connected to the computer-readable medium. In some embodiments, the processor is configured to execute instructions for implementing the methods described herein. The system can further include means for user input and output, such as a keyboard, a monitor, and a mouse.

[0181] In some embodiments, the systems described herein can be configured to access databases, e.g., local or online (e.g., cloud) Exemplary databases include protein structure databases, protein sequence databases, homology databases, nucleic acid sequence databases, etc.

[0182] When the methods described herein are performed, the system can further include information obtained by performing the methods described herein. For example, when the methods described herein are performed, the system can include predicted results from evaluating the scaffold protein(s) and / or engineered scaffold protein(s). The system can include the amino acid sequence of the engineered scaffold protein generated from performing the methods described herein. Additionally, the system can include data and / or results generated from the in silico, in vitro, and / or in vivo assays and / or machine learning models described herein. Such a system can include a means for transferring information obtained by the methods described herein. In some cases, the system can include a means for transmitting information obtained by the methods described herein to an external database (e.g., a local database or an online database).

[0183] Epitope Masking Unit In some embodiments, the compositions and methods provided herein further comprise an epitope masking unit or its use. The epitope masking unit provided herein can at least partially shield the binding domain of the engineered scaffold protein, thereby masking the binding epitope contained on the surface of the binding domain. In certain embodiments, the binding domain comprises one or more amino acid changes compared to the corresponding WT, and in such embodiments, the epitope masking unit provided herein can prevent the one or more amino acid changes from being recognized by the immune system. Such epitope masking units can also function as inhibitors of autoimmunity.

[0184] In some embodiments, epitope masking unit can be any peptide that can non-covalently interact or bind with the binding domain described herein.In some embodiments, epitope masking unit is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% antisense to one or more amino acids of the binding domain of engineered scaffold protein, as determined by corresponding sense-antisense amino acid pairing.In some embodiments, epitope masking unit provided herein can be found in nature together with the WT counterpart of engineered scaffold protein.When using the MHC class II scaffold protein from which the engineered scaffold protein provided herein is derived, an example is the use of CLIP peptide or its variant.

[0185] In some embodiments, the epitope masking unit described herein can also function to minimize off-target binding.In some embodiments, the engineered scaffold proteins provided herein are not bound by target peptides.In such embodiments, the epitope masking unit described herein can bind to the binding domain so that only peptides with strong binding affinity can replace the epitope masking unit.In such embodiments, other off-target peptides need to compete with the epitope masking unit to bind to the binding domain.

[0186] In some embodiments, the epitope masking unit may also function to stabilize the accepted conformation of the engineered scaffold proteins provided herein, such that the engineered scaffold proteins maintain a conformation that is more suitable for binding to the disordered regions described herein.

[0187] The epitope masking unit provided herein can be associated with the engineered scaffold protein described herein.The epitope masking unit can be attached to the engineered scaffold protein.For example, the epitope masking unit can be fused, linked, or covalently or non-covalently bound to the engineered scaffold protein.The epitope masking unit can be linked by a linker described herein, such as a self-cleavable linker or a proteolytically cleavable linker.The epitope masking unit can be bound to a proximal cysteine ​​or other proximal residue.The epitope masking unit can be attached to the C-terminus, N-terminus, or mid-loop of the engineered scaffold protein.

[0188] Fusion Partners In some embodiments, the compositions and methods provided herein further comprise a fusion partner or the use thereof. Fusion proteins comprising an engineered scaffold protein described herein and a fusion partner are also provided herein. In some embodiments, fusion proteins comprising multiple engineered scaffold proteins described herein are multispecific (e.g., bispecific). Such multispecific fusion proteins can comprise two, three, four, or more engineered scaffold proteins and one, two, or more fusion partners.

[0189] Fusion partners provided herein include one or more of the following: a protein or functional fragment thereof, an enzyme or functional fragment thereof, an antibody or functional fragment thereof, a compound or molecule that increases the half-life of an engineered scaffold protein, a cytotoxic agent, a therapeutic agent, an immunological agent, a detection agent, two or more of the foregoing, or any combination thereof. Examples of fusion partners comprising a protein or functional fragment thereof include human serum albumin (HSA) or a domain thereof (e.g., an HSA-binding domain), fibrinogen or a domain thereof (e.g., a fibrinogen-binding domain), complement component 1q (C1q), transferrin or a domain thereof (e.g., a transferrin-binding domain), a cell-penetrating peptide (CPP), etc. Examples of fusion partners comprising an enzyme or functional fragment thereof include ubiquitin ligase, trypsin, kinase, phosphatase, chaperone, etc. Examples of fusion partners comprising an antibody or functional fragment thereof include the Fc region of an antibody. Examples of fusion partners containing compounds or molecules that increase the half-life of engineered scaffold proteins include polymers (e.g., polyethylene glycol or PEGylation), carbohydrates, post-translational modifications (e.g., N-terminal glycosylation and / or polymer mimetics), HSA-binding domains, variant Fc domains, transferrin-binding domains, fibrinogen-binding domains, etc. See, for example, examples of fusion partners containing cytotoxic agents include the Fc region of an antibody, complement component 1q, chemotherapeutic agents, etc. Examples of fusion partners containing therapeutic agents or therapeutic partners described herein include anti-cancer drugs, anti-inflammatory drugs, antibacterial drugs, antiviral drugs, cytokines, toxins, enzymes, neuroprotective agents, soluble factor capture agents, etc. Examples of fusion partners containing immunological agents include foreign antigens, etc. Examples of fusion partners containing detection agents include fluorescent molecules, radioisotopes, reporter molecules, detectable signals, etc.Other examples of suitable fusion partners are described in Zaman R, et. al., Current strategies in extending half-lives of therapeutic proteins. J Control Release. 2019 May 10;301:176-189. doi:10.1016 / j.jconrel.2019.02.016. Epub 2019 Mar 5. PMID: 30849445, and Silver, et. al., Engineered antibody fusion proteins for targeted disease therapy, Trends in Pharmacological Sciences, Volume 42, Issue 12, 2021, Pages 1064-1081, ISSN 0165-6147, doi.org / 10.1016 / j.tips.2021.09.009. (sciencedirect.com / science / article / pii / S0165614721001899).

[0190] Exemplary fusion partners are listed in Table 10.

[0191] Binding of the engineered scaffold protein or its fusion protein can modify the specific biology / biochemistry of the target peptide, the intracellular microenvironment containing the target peptide, the cell expressing the target peptide, or the extracellular environment in which the target peptide is localized.

[0192] In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein act as antagonists or inhibitors upon binding to a target protein. The term "inhibitor," as used herein in connection with an engineered scaffold protein or fusion protein thereof, refers to a protein that can interfere with, limit, or prevent the activity of the target protein upon binding to the target protein. Such engineered scaffold proteins or fusion proteins thereof can function in various therapeutic modalities described herein. In some embodiments, the engineered scaffold protein or fusion protein thereof, upon binding to the target protein, acts as an agent for inducing degradation of the target protein by ubiquitination of the target protein. In some embodiments, the engineered scaffold protein or fusion protein thereof is conjugated to a drug, which, upon binding to the target protein, delivers the drug to cells expressing the target protein. Also provided herein, in some embodiments, is a fusion protein comprising the engineered scaffold protein described herein and an Fc region of an antibody, which promotes antibody-dependent cellular cytotoxicity (ADCC) upon binding to a target protein expressed by a cell. Also provided herein in some embodiments is an engineered scaffold protein described herein, or a fusion protein thereof, that prevents ADCC upon binding to a target protein expressed by a cell. Also provided herein in some embodiments is a fusion protein comprising an engineered scaffold protein described herein and a fusion partner capable of binding complement component 1q (C1q), that promotes complement-dependent cytotoxicity (CDC) upon binding to a target protein expressed by a cell. Also provided herein in some embodiments is an engineered scaffold protein described herein, or a fusion protein thereof, that prevents CDC upon binding to a target protein expressed by a cell. Also provided herein in some embodiments is an engineered scaffold protein described herein, or a fusion protein thereof, that results in the presentation of an antigen on an antigen-presenting cell, thereby generating an immune response.Such engineered scaffold proteins or fusion proteins thereof can be used as infectious disease vaccines, neurodegenerative vaccines, or cancer neoepitope vaccines.

[0193] In some embodiments, the engineered scaffold protein or fusion protein thereof provides a target-specific marker for use in detecting diagnostic markers of disease. This includes generating or using panels of engineered scaffold proteins or fusion proteins thereof for autoimmune diseases, various tumor indications, neurodegenerative diseases, etc. In some embodiments, the engineered scaffold protein or fusion protein thereof provides a target-specific binding agent and / or reagent for in situ and alternative immunofluorescence detection. In some embodiments, the engineered scaffold protein or fusion protein thereof provides a target-specific binding agent and / or reagent for protein fingerprinting and sequencing.

[0194] In some embodiments, the engineered scaffold proteins or fusion proteins thereof provided herein are inhibitors or contain inhibitory activity, and when bound to a target protein, the engineered scaffold proteins or fusion proteins thereof can function as inhibitors of the target protein, preventing the biological activity of the target protein from occurring. Such biological activities can include protein-protein interactions, protein-nucleic acid interactions, protein isomerization, protein post-translational modifications (e.g., phosphorylation, glycosylation, acetylation, ubiquitination, proteolytic cleavage, etc.), protein refolding, and protein aggregation. Biological activities can also include mechanisms by which the binding of one interacting partner can be inhibited, thereby promoting more efficient binding of a different interacting partner and eliciting a different biological activity. In this scenario, the therapeutic target can be a protein other than the target protein, either in the extracellular matrix or the intracellular environment. Interfering with biological activity can impede cell growth, for example, by disrupting certain signaling pathways involved in apoptosis. By binding to targets that interfere with immune cell activity, the immune system itself can destroy diseased cells (e.g., cancer cells) or pathogenic cells.

[0195] In some embodiments, the engineered scaffold proteins or fusion proteins thereof provided herein comprise an activity provided by a fusion partner described herein.

[0196] In some embodiments, the engineered scaffold protein is fused with a trypsin enzyme and selectively binds to specific disordered regions on the target protein. Upon binding, any lysine or arginine residues near the binding interface of the target protein are cleaved by trypsin, thereby destroying the target protein. In some embodiments, the engineered scaffold protein is fused with a kinase or phosphatase and selectively binds to specific disordered regions on the target protein. Upon binding, the kinase or phosphatase then preferentially phosphorylates (in the case of kinases) or dephosphorylates (in the case of phosphatases) residues near the binding site. In some embodiments, the engineered scaffold protein is fused with an E3 ligase and selectively binds to specific disordered regions on the target protein. Upon binding, the E3 ligase then ubiquitinates the target protein near the binding site, thereby targeting the protein of interest for degradation via the proteasome. In some embodiments, the engineered scaffold protein is fused to the Fc region of an antibody to act as a fusion antibody molecule, where the engineered scaffold protein can serve the purpose of an antibody Fab domain that specifically binds to a known epitope of the antigen (i.e., the disordered region that the binding agent specifically recognizes). In each of the embodiments, the engineered scaffold protein acts as a homing agent that specifically targets the protein of interest and effects a biological change or activity on the protein of interest through the action of the fusion partner.

[0197] In some embodiments, the present invention provides a fusion protein comprising an engineered scaffold protein described herein and a fusion partner, wherein the fusion partner is a chaperone protein. Here, the engineered scaffold protein targets a specific disordered region of a misfolded protein, and the chaperone molecule acts inducibly to refold the misfolded protein. For example, in emphysema, α1-antitrypsin is a protease inhibitor that is misfolded and accumulates in hepatocytes. α1-antitrypsin has several disordered loops in its predicted structure, one of which is ALVNYIFFKGK (SEQ ID NO: 925). By designing a binder for this disordered region and fusing it with a chaperone enzyme (e.g., Hsp70 or Hsp90), the misfolded α1-antitrypsin can be specifically targeted by the binder, and the chaperone can correctly fold the protein.

[0198] In some embodiments, the engineered scaffold proteins provided herein are generic scaffolds or generic scaffold proteins, which comprise a binding framework encompassing a structural basis for designing and engineering multiple different artificial binding sites. The binding sites of such scaffold proteins can be loops or rigid structural domains in the scaffold protein. Such generic scaffold proteins may consist of a single polypeptide chain. Additionally, generic scaffold proteins may have inherent conformational and thermodynamic stability and may be humanizable. Generic scaffold proteins can also be engineered to bind with high affinity and specificity to multiple different disordered regions in different proteins. In other words, generic scaffold proteins can be modified to yield different engineered scaffold proteins with different binding properties (e.g., sequence specificity and / or affinity).

[0199] The half-life of the engineered scaffold proteins or fusion proteins thereof disclosed herein can be extended by several techniques. In some embodiments, the half-life of the engineered scaffold proteins or fusion proteins thereof disclosed herein can be extended by fusion with a compound or molecule described herein. For example, the half-life of the engineered scaffold protein or fusion protein thereof can be extended by chemically conjugating it to a polymer, carbohydrate, post-translational modification including N-terminal glycosylation, and / or a polymer mimetic, which can increase the overall protein size, thereby promoting reduced renal clearance and extending the bioavailability of the biologic or protein. Such chemical modifications can also help prevent the action of other molecules or proteins that would otherwise degrade the engineered scaffold or fusion protein thereof.

[0200] Another strategy to improve half-life involves fusing the engineered scaffold or its fusion protein with an HSA-binding domain, a variant Fc domain, a transferrin-binding domain, or a fibrinogen domain. Fusion with any of these domains can increase the size of the protein, thereby reducing the rate of renal clearance. Additionally, all of these fusion partners can bind to the Fc-Rn receptor or CTLA receptor, allowing for receptor-mediated endocytosis and thus increasing half-life. See, for example, Al-Qahatani et al. (2019), Biomedicine & Pharmacotherapy; Zaman et al. (2019), Journal of Controlled Release. Half-life can also be increased by fusing the engineered scaffold or its fusion protein with an antibody ScFv region or by PEGylation.

[0201] Multiple engineered scaffold proteins and / or fusion partners can be fused or linked together to create multispecific drugs (e.g., fusion proteins). The multispecific scaffold construct described herein can refer to a construct encoding or containing an engineered scaffold protein described herein or a fusion protein thereof that can target multiple proteins and inhibit all of the target proteins as an antagonist, or function as an agonist for all of the target proteins, or be an antagonist for a subset of the target proteins and an agonist for a subset of the target proteins. Protein domains can be fused or linked together to generate an engineered scaffold protein. For example, one or more binding units, hinge units, immunoglobulin units, epitope masking units, and / or linking units can be fused or linked together. In another example, one or more alpha helices, beta sheets, immunoglobulin units, or combinations thereof can be fused or linked together.

[0202] Linker In some embodiments, the engineered scaffold protein described herein comprises one or more linkers.The linkers disclosed herein can be flexible, cleavable, or rigid depending on the nature of the application.Additional linkers can be used to adjust solubility, increase expression, improve biological activity, enable targeting, or change Pk.See, for example, Chen et al., Adv Drug Deliv Rev., 65(10):1357-69(2013).

[0203] In some embodiments, the engineered scaffold proteins provided herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more linkers. In some embodiments, the engineered scaffold proteins provided herein do not comprise any linkers. In some embodiments, the peptide linker has a length of 3 to 25 amino acids. In some embodiments, the linking units described herein can be substituted for the linkers provided herein.

[0204] In some embodiments, the linker may be a peptide linker or a non-peptide linker. In some embodiments, the peptide linker has a length of 1 to 30 amino acids. In some embodiments, the peptide linker has a length of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids or more.

[0205] The protein domains can be linked together by a short peptide linker, which can be any one of several of the following, but is not limited to: LE; (GGS)nn=1,2,3,4,5; (GGGGS)nn=1,2,3,4,5-(SEQ ID NO:973); (SSG)nn=1,2,3,4,5; GGGGGG (SEQ ID NO:974); GGGGGGGG (SEQ ID NO:975); and peptide 1-nucleic acid-peptide 2. In some embodiments, peptide 1 is any peptide sequence of 5 or more amino acids with a functional group at the N-terminus. In some embodiments, peptide 2 is any peptide sequence of 5 or more amino acids with a functional group at the C-terminus of the peptide. In some embodiments, the two peptides are conjugated to each other by a nucleic acid.

[0206] The constructs disclosed herein can be assembled in multiple ways. In some embodiments, the construct is N-terminus-Binder1-Linker1-Binder2-Linker2-Binder3-Linker3....-Linker(n-1)-Binder(n)-C-terminus, where "N-terminus" refers to the N-terminus of the biologic (e.g., a therapeutic protein), "C-terminus" refers to the C-terminus of the molecule, and "binder" refers to an engineered scaffold protein or other binding protein, provided that at least one engineered scaffold protein is present in the molecule. In some embodiments, a fusion protein containing two engineered scaffold proteins is bispecific. Bispecificity is a specific example of two engineered scaffold proteins linked together in the construct. Each linker can be any one of the linkers disclosed herein, and any combination of linkers can be used to fuse the binder moieties together.

[0207] In some embodiments, a multispecific scaffold construct or its use is described herein. The multispecific scaffold construct can target multiple proteins and can inhibit all of the target proteins as an antagonist, can function as an agonist for all of the target proteins, or can be an antagonist for a subset of the target proteins and an agonist for a subset of the target proteins. A single domain or a subset of domains in the multispecific scaffold construct can also be used as a means of homing to the target cell or target protein, and other domains in the therapeutic agent can function as inhibitors or agonists, or a combination of inhibitors and agonists.

[0208] Multispecific scaffold constructs can disrupt protein-protein interactions by simultaneously binding to multiple partners in a protein interaction network, disrupting important cellular processes necessary for cell growth and survival. Multispecific therapeutics can also function to assemble together several proteins that may not be able to assemble on their own, acting as a hub to facilitate protein-protein interactions between all binding partners.

[0209] Exemplary linkers are listed in Table 9.

[0210] vector The compositions and methods described herein include one or more vectors or their use. In some embodiments, one or more vectors used herein comprise one or more nucleotide sequences encoding one or more components of a composition or method described herein. In some embodiments, one or more vectors used herein are conjugated or otherwise attached to one or more components of a composition or method described herein. In some embodiments, one or more components comprise one or more engineered scaffold proteins, fusion partners, linkers, fusion proteins, targeting peptides, or functional fragments thereof. In some embodiments in which more than one vector is used, the compositions and methods described herein can include a library of vectors, each vector encoding or conjugated to one or more components of a composition or method described herein. In some embodiments, the components described herein are encoded by or conjugated to the same vector, or each component is encoded by or conjugated to a different vector, or a combination thereof.

[0211] In some embodiments, the vectors described herein comprise or encode one or more regulatory elements. Such regulatory elements can be operably linked to a nucleotide sequence encoding an engineered scaffold protein, a fusion partner, a linker, a fusion protein, a target peptide, or a functional fragment thereof, or two or more of the foregoing. Regulatory elements can include transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and proteolysis signals, that enable and / or regulate the transcription of non-coding or coding sequences and / or regulate the translation of encoded polypeptides. In some embodiments, the vectors comprise or encode one or more additional elements, such as, for example, an origin of replication, antibiotic resistance (or a nucleic acid encoding same), a tag (or a nucleic acid encoding same), and a selectable marker. In some embodiments, the vectors comprise or encode one or more elements, such as, for example, a ribosome binding site and an RNA splice site. Generally, the vectors provided herein comprise at least one promoter or a combination of promoters that drive the expression or transcription of an engineered scaffold protein, a fusion partner, a fusion protein, a target peptide, or a functional fragment thereof, as described herein.

[0212] The vector can be an expression vector. The vector can be a viral vector, such as an AAV vector or a lentiviral vector, or a non-viral vector, such as a lipid, a lipid particle, a cell-penetrating peptide, or an mRNA.

[0213] The engineered scaffolds or fusion proteins thereof disclosed herein may be used for therapeutic purposes by delivering the engineered scaffolds or fusion proteins thereof to target cells. Intracellular delivery of such therapeutic agents may be by AAV vectors, lentiviral vectors, cell-penetrating peptides, or as mRNA.

[0214] The engineered scaffold protein or fusion protein thereof may be used for a polypeptide display library. In some embodiments, the polypeptide display library provided herein comprises an engineered scaffold protein or fusion thereof expressed and displayed by a vector described herein. In some embodiments, the engineered scaffold protein or fusion protein or fusion thereof is included in an expression vector and delivered to a host cell for expression. Thus, in some embodiments, host cells comprising the engineered scaffold protein or fusion protein described herein are provided herein. In some embodiments, the polypeptide display library provided herein displays the engineered scaffold protein or fusion thereof on the surface of a virus or yeast, or displays the engineered scaffold protein or fusion thereof displayed as a ribosome or RNA-conjugated protein molecule.

[0215] Also provided herein in some embodiments is an isolated nucleic acid molecule that encodes an engineered scaffold protein described herein, a fusion thereof, one or more components of the following, or a combination thereof. In some embodiments, an expression vector can be operably linked to an isolated nucleic acid molecule provided herein. Thus, provided herein are compositions or methods comprising an isolated nucleic acid molecule that encodes an engineered scaffold protein described herein, a fusion thereof, one or more components of the following, or a combination thereof, and in some embodiments, the isolated nucleic acid molecule operably linked to an expression vector.

[0216] Target peptide Disclosed herein are compositions and methods comprising target peptides or their uses. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein interact with or bind to the target peptide. In some embodiments, interacting with or binding to the target peptide results in the detection of the target peptide or the induction of a biological or therapeutic activity. Thus, compositions and methods for detecting the target peptide or inducing a biological or therapeutic effect are also disclosed herein.

[0217] The target peptides described herein can be part of a polypeptide, a longer protein, or a protein complex. Thus, reference to a target peptide contained in or composed with a polypeptide, protein, or protein complex also refers to the target polypeptide, target protein, and / or target protein complex. In some embodiments, reference to a target peptide also refers to a nucleic acid (i.e., a DNA or RNA molecule) that encodes the target peptide. Thus, in some embodiments, the compositions and methods described herein include a nucleic acid that encodes the target peptide described herein. In some embodiments, a target peptide may be referred to herein as a target ligand.

[0218] In some embodiments, the target peptide has a length of 2 to 50, 3 to 40, or 4 to 30 amino acids. In some embodiments, the target peptide has a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, or 50 amino acids. In some embodiments, the target peptide has at least 4, at least 15, or at least 30 amino acids.

[0219] In some embodiments, the target peptide is contained in a polypeptide, protein, or protein complex. In some embodiments, the length of one protein in the polypeptide, protein, or protein complex comprising the target peptide may be greater than about 30 amino acids. In some embodiments, the target peptide is contained in a polypeptide, protein, or protein complex, and the length of one protein in the polypeptide, protein, or protein complex is greater than about 30 amino acids. In some embodiments, the length of one protein in the polypeptide, protein, or protein complex comprising the target peptide is greater than about 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 130 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 220 amino acids, 230 amino acids, 240 amino acids. 250 amino acids, 260 amino acids, 270 amino acids, 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, 330 amino acids, 340 amino acids, 350 amino acids, 360 amino acids, 370 amino acids, 380 amino acids, 390 amino acids, 400 amino acids, 410 amino acids, 420 amino acids, 430 amino acids, 440 amino acids, 450 amino acids, 460 amino acids, 470 amino acids, 480 amino acids, 490 amino acids, 500 amino acids or longer.

[0220] In some embodiments, the weight of a polypeptide, protein, or single protein of a protein complex comprising a target peptide may be greater than about 100 daltons. In some embodiments, the target peptide is comprised in a polypeptide, protein, or protein complex, and the weight of a single protein of the polypeptide, protein, or protein complex is greater than about 100 daltons, 1000 daltons, 2000 daltons, 4000 daltons, 6000 daltons, 8000 daltons, 10000 daltons, 12000 daltons, 14000 daltons, 16000 daltons, 18000 daltons, 20000 daltons, 22000 daltons, 24000 daltons, 26000 daltons, 28000 daltons, 30000 daltons, 32000 daltons, 34000 daltons, 36000 daltons, 38000 daltons, 40000 daltons, 42000 daltons. , 44,000 Daltons, 46,000 Daltons, 48,000 Daltons, 50,000 Daltons, 52,000 Daltons, 54,000 Daltons, 56,000 Daltons, 58,000 Daltons, 60,000 Daltons, 62,000 Daltons, 64,000 Daltons, 66,000 Daltons, 68,000 Daltons, 70,000 Daltons, 72,000 Daltons, 74,000 Daltons, 76,000 Daltons, 78,000 Daltons, 80,000 Daltons, 82,000 Daltons, 84,000 Daltons, 86,000 Daltons, 88,000 Daltons, 90,000 Daltons, 92,000 Daltons, 94,000 Daltons, 96,000 Daltons, 98,000 Daltons, 100,000 Daltons or greater.

[0221] In some embodiments, where the target peptide is contained in a polypeptide, protein, or protein complex, the engineered scaffold proteins described herein can be engineered to be multispecific (e.g., bispecific or trispecific) to bind to multiple regions of the polypeptide, protein, or protein complex. The regions can be disordered regions, ordered regions, or a combination thereof.

[0222] In some embodiments, the target peptides described herein are contained in cells. In some embodiments, the target peptides described herein are contained in a population of cells. In some embodiments, the target peptides described herein are contained in naturally occurring cells, eukaryotic cells, prokaryotic cells, plant cells, fungal cells, animal cells, invertebrate cells, fly cells, vertebrate cells, mammalian cells, primate cells, non-human primate cells, human cells, living cells, non-living cells, modified cells, derived cells, non-naturally occurring cells, or any combination thereof. In some embodiments, the cells comprising the target peptides described herein are contained in eukaryotic cells. In some embodiments, the target peptides described herein are contained in a population of naturally occurring cells, eukaryotic cells, prokaryotic cells, plant cells, fungal cells, animal cells, invertebrate cells, fly cells, vertebrate cells, mammalian cells, primate cells, non-human primate cells, human cells, living cells, non-living cells, modified cells, derived cells, non-naturally occurring cells, or any combination thereof. In some embodiments, the cells comprising the target peptides described herein are contained in a population of eukaryotic cells.

[0223] In some embodiments, the target peptides described herein are isolated from cells. In some embodiments, the target peptides described herein are isolated from any one of naturally occurring cells, eukaryotic cells, prokaryotic cells, plant cells, fungal cells, animal cells, invertebrate cells, fly cells, vertebrate cells, mammalian cells, primate cells, non-human primate cells, human cells, living cells, non-living cells, modified cells, derived cells, and non-naturally occurring cells. In some embodiments, the target peptides are isolated from a population of cells. In some embodiments, the target peptides described herein are isolated from a population of naturally occurring cells, eukaryotic cells, prokaryotic cells, plant cells, fungal cells, animal cells, invertebrate cells, fly cells, vertebrate cells, mammalian cells, primate cells, non-human primate cells, human cells, living cells, non-living cells, modified cells, derived cells, non-naturally occurring cells, or any combination thereof. In some embodiments, the target peptides described herein are contained in a sample. In some embodiments, the target peptides described herein are contained in a sample obtained or harvested from an organism. In some embodiments, the target peptides described herein are contained in a sample obtained or harvested from a eukaryote, a prokaryote, a plant, a fungal organism, a bacterium, a virus, an animal, an invertebrate, a fly, a vertebrate, a mammal, a mouse, a primate, a non-human primate, or a human.

[0224] In some embodiments, the target peptide described herein, or a cell or organism containing the target peptide, is included in a system for detecting the target peptide, such as a kit described herein. In some embodiments, the target peptide described herein is included in a device for detecting the target peptide. In some embodiments, the target peptide is naturally occurring or includes one or more modifications, such as modifications for use in the systems or methods described herein. Such modifications include fusion or conjugation with a detection agent described herein.

[0225] In some embodiments, the target peptides described herein are included in or are extracellular proteins. In some embodiments, the target peptides described herein are membrane proteins. In some embodiments, the target peptides described herein are included in or are G protein-coupled receptors (GPCRs), ion channels, or secreted proteins. In some embodiments, the target peptides described herein are included in or are any of the proteins or portions thereof listed in Table 7.

[0226] In some embodiments, the target peptides described herein are associated with a disease or disorder. In some embodiments, the target peptides described herein are encoded by a nucleic acid associated with a disease or disorder. In some embodiments, the target peptides described herein are contained in a cell associated with a disease or disorder. In some embodiments, the target peptides described herein are contained in an organism suffering from a disease or disorder. In some embodiments, the disease or disorder described herein is a genetic disease or disorder, a pathogenic disease or disorder, an infectious disease or disorder, a transmissible disease or disorder, an immunological disease or disorder, a mutagenic disease or disorder, or any combination thereof. In some embodiments, the disease or disorder described herein is associated with any of the target proteins listed in Table 7. Methods of treating, preventing, or inhibiting a disease or disorder are also disclosed herein.

[0227] In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can interact with or bind to a disordered region of a target peptide. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein interact with or bind to a linear epitope present in a disordered region of a target peptide. In some embodiments, the target peptide may be an intrinsically disordered peptide. In some embodiments, the target peptide may comprise a regular structure, but still comprise a disordered region.

[0228] In some embodiments, the engineered scaffold proteins or fusion proteins thereof provided herein can interact with or bind to a regular region of a target peptide. In some embodiments, the engineered scaffold proteins or fusion proteins thereof provided herein can interact with or bind to a regular region and an irregular region of a target peptide, either in the same binding domain or in different binding domains. Thus, the engineered scaffold proteins or fusion proteins described herein that can be engineered to bind to irregular regions as described herein can also be engineered to bind to regular regions.

[0229] irregular area In some embodiments, the engineered scaffold proteins described herein can be engineered to interact with or bind to the disordered regions of the target peptides described herein. In some embodiments, the disordered regions can be fully disordered or partially disordered. Once the target peptide is selected, the disordered regions can be determined from Uniprot or another sequencing database, and disordered epitopes comprising the complete region or a subset of that disordered region can be used to develop engineered scaffold proteins according to any of the methods and examples disclosed herein.

[0230] In some embodiments, the disordered region of the target peptide can adopt an extended conformation to which the engineered scaffold proteins described herein can bind. In some embodiments, the engineered scaffold proteins described herein are engineered to interact with or bind to the disordered region of the target peptide having an extended conformation. In some embodiments, the extended conformation of the disordered region is a linear conformation or sequence.

[0231] In some embodiments, the irregular region is comprised in a loop, C-terminal tail, or N-terminal tail of a target peptide described herein. In some embodiments, the engineered scaffold proteins described herein are engineered to interact with or bind to a loop, C-terminal tail, or N-terminal tail of a target peptide described herein. In some embodiments, the engineered scaffold proteins described herein are engineered to interact with or bind to a irregular region comprised in a loop, C-terminal tail, or N-terminal tail of a target peptide described herein. In some embodiments, the engineered scaffold proteins described herein are engineered to interact with or bind to a linear sequence of a irregular region comprised in a loop, C-terminal tail, or N-terminal tail of a target peptide described herein.

[0232] The disordered regions of the target peptides described herein may have known biological functions, hi some embodiments, the engineered scaffold proteins described herein are designed to bind to the target peptide and thereby induce, alter, or inhibit the biological function of the disordered region.

[0233] In some embodiments, the disordered regions of the target peptides described herein can function as recognition sites for enzyme active sites or as ligand sites recognized by the binding surfaces of one or more protein partners. As described herein, protein partner(s) refer to polypeptides capable of recognizing and interacting with the ligand site of a target peptide. In some embodiments, the engineered scaffold proteins described herein are engineered to interact with or bind to disordered regions that function as recognition sites for enzyme active sites or as ligand sites recognized by the binding surfaces of one or more protein partners. In some embodiments, binding of the engineered scaffold protein to a disordered region that functions as a recognition site can induce structural modifications, proteolytic cleavage, and post-translational modification removal or addition of the target peptide, as described in more detail herein. In some embodiments, binding of the engineered scaffold protein to a disordered region that functions as a ligand site can induce complexation promotion, docking, and targeting or transport of the protein partner(s), as described in more detail herein. In some embodiments, binding of the engineered scaffold protein to the target peptide can activate the disordered region that functions as a recognition or ligand site, thereby inducing the above-mentioned activity. In some embodiments, binding of the engineered scaffold protein to the target peptide can mask the disordered regions that function as recognition or ligand sites, thereby inhibiting the aforementioned activity.

[0234] In some embodiments, an irregular region of a target peptide can function as a promoter of protein-protein interaction. In some embodiments, the engineered scaffold proteins described herein are engineered to interact with or bind to an irregular region that functions as a promoter of protein-protein interaction. In some embodiments, such an irregular region comprises a preformed structural element (PSE), a molecular recognition feature (MoRF), a molecular recognition (MoRE), or a prestructured motif (PreSMo). In some embodiments, an irregular region comprising a MoRF undergoes a disordered-to-order transition (i.e., folds upon binding) upon binding to an interaction partner. The interaction partner can be any protein that binds to an irregular region that functions as a promoter of protein-protein interaction. In some embodiments, an irregular region comprising a PSE or MoRF can function as the initial contact point of an interaction event, leading to conformational selection and folding of the preformed element. In some embodiments, binding of the engineered scaffold protein to a target peptide can activate the irregular region that functions as a promoter of protein-protein interaction, thereby inducing the above-mentioned activity. In some embodiments, binding of the engineered scaffold protein to the target peptide can mask the disordered regions that function as facilitators of protein-protein interactions, thereby inhibiting the aforementioned activities.

[0235] In some embodiments, the disordered region of the target peptide can function as a DNA-binding region, an RNA-binding region, or a protein-binding region. In some embodiments, the binding of the engineered scaffold protein to the target peptide can activate the disordered region that functions as a DNA-, RNA-, or protein-binding region, thereby inducing the above-mentioned activity. In some embodiments, the binding of the engineered scaffold protein to the target peptide can mask the disordered region that functions as a DNA-, RNA-, or protein-binding region, thereby inhibiting the above-mentioned activity.

[0236] In some embodiments, the disordered region comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, or at least 50 consecutive amino acids of the target peptide, including the complete sequence or a subset of the listed sequences.

[0237] Based on the selected target peptide sequence, disordered regions can be determined from Uniprot, and disordered epitopes comprising the complete region or a subset of that disordered region can be used for the development of engineered scaffold proteins according to any of the methods and examples disclosed herein. Such engineered scaffold protein development can also be utilized for any of the applications disclosed herein, including vaccine development, therapeutic drug development, immune modulation / suppression, detection, and diagnostics.

[0238] In embodiments in which two or more engineered scaffold proteins or fusion proteins thereof are used, such engineered scaffold proteins or fusion proteins may recognize the same linear epitope or distinct linear epitopes.

[0239] Non-limiting examples of linear epitopes and disordered regions of target peptides are listed in Table 7.

[0240] Pharmaceutical Composition In certain embodiments, disclosed herein are pharmaceutical compositions comprising an engineered scaffold protein described herein and a carrier thereof for administration to a subject.

[0241] In certain embodiments, pharmaceutically acceptable compositions comprise a therapeutically effective amount of one or more engineered scaffold proteins formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. In some embodiments, when the engineered scaffold proteins herein are administered to humans and animals as pharmaceuticals, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1-99% or 10-30% engineered scaffold protein in combination with a pharmaceutically acceptable carrier.

[0242] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously, for example, using a standard needle and syringe or a pen-type delivery device. Injectable preparations can include dosage forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. Injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying the engineered scaffold protein herein in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, and the like, which can be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), and the like. Oily media include, for example, sesame oil, soybean oil, and the like, which can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, and the like. The injection thus prepared can be filled into suitable ampoules.

[0243] The compositions of the present disclosure may be in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or liquids. The amount of engineered scaffold protein disclosed herein contained may be about 5 to about 500 mg per dosage form in a unit dose. In one embodiment, the engineered scaffold protein may be contained in an amount of about 5 to about 100 mg, for example, in a parenteral dosage form. In other embodiments, the engineered scaffold protein may be contained in an amount of about 10 to about 250 mg in other dosage forms.

[0244] For example, oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gel capsules, and caplets can be used as solid dosage forms. These can be prepared, for example, by mixing the engineered scaffold protein with at least one additive, such as starch or other additives. Suitable additives include sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Optionally, oral dosage forms can contain other ingredients to aid administration, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens or sorbic acid, or antioxidants such as ascorbic acid, tocopherol, or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, or flavoring agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[0245] Liquid dosage forms for oral administration can be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inert diluent such as water. In some embodiments, pharmaceutical formulations and medicaments can be prepared as liquid suspensions or aqueous solutions, for example, using a sterile liquid, such as, but not limited to, oil, water, alcohol, or any combination thereof. In some embodiments, pharmaceutical compositions can be prepared in lyophilized form. Lyophilized preparations can contain cryoprotectants known in the art. The term "cryoprotectant," as used herein, generally includes agents that provide proteins with stability against stress caused by freezing. Examples of cryoprotectants include polyols, such as mannitol, sugars, such as sucrose, and surfactants, such as polysorbates, poloxamers, or polyethylene glycols. Cryoprotectants also contribute to the tonicity of the formulation. Pharmaceutically suitable surfactants, suspending agents, and emulsifying agents can be added for oral or parenteral administration.

[0246] As mentioned above, suspensions can contain oil.Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil.Suspension preparations can also contain fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides.Suspension formulations can contain alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol.Ethers, such as, but not limited to, poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water can also be used in suspension formulations.

[0247] For nasal administration, the pharmaceutical formulation and medicament may be a spray or aerosol containing a suitable solvent(s) and optionally other compounds, such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH control agents, surfactants, bioavailability modifiers, or any combination thereof. Propellants for aerosol formulations may include compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low-boiling point solvents.

[0248] Injectable dosage forms generally include aqueous or oily suspensions, which can be prepared using suitable dispersants or wetting agents and suspending agents.Injectable forms can be in the form of a solution phase or suspension, which can be prepared using a solvent or diluent.Acceptable solvents or vehicles include sterile water, Ringer's solution, or isotonic saline solution.Alternatively, sterile oils can be used as solvents or suspending agents.In some embodiments, oils or fatty acids are non-volatile, such as natural or synthetic oils, fatty acids, monotriglycerides, ditriglycerides, or triglycerides.

[0249] For injection, pharmaceutical formulation and / or medicament may be powder suitable for reconstitution with appropriate solution as described above.Examples of these include, but are not limited to, freeze-dried, rotary-dried or spray-dried powder, amorphous powder, granule, precipitate, or particulate.For injection, formulation may optionally contain stabilizer, pH control agent, surfactant, bioavailability adjuster, or any combination thereof.

[0250] For rectal administration, pharmaceutical formulations and medicaments may be in the form of suppositories, ointments, enemas, tablets, or creams for compound release in the intestines, sigmoid colon, and / or rectum. Rectal suppositories are prepared by mixing one or more compounds herein with an acceptable vehicle, such as cocoa butter or polyethylene glycol, which exists in a solid phase at normal storage temperatures and in a liquid phase at temperatures suitable for drug release in the body, such as the rectum. Oils can also be used to prepare soft gelatin-type formulations and suppositories. Water, saline, dextrose and related sugar aqueous solutions, and glycerol can be used to prepare suspension formulations, which may also contain suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylcellulose, or carboxymethylcellulose, as well as buffers and preservatives.

[0251] The concentration of engineered scaffold protein in these compositions can vary widely, for example, from less than about 10% by weight, at least about 25% by weight, up to 75% or 90% by weight, and is selected primarily based on fluid volume, viscosity, etc., according to the particular mode of administration selected.

[0252] In some embodiments, pharmaceutical compositions comprising the engineered scaffold proteins described herein can be formulated using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate processing of the active compounds into pharmaceutically usable preparations. Appropriate formulations depend on the route of administration selected.

[0253] Pharmaceutical compositions are optionally manufactured by means of, for example only, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, embedding, or compressing processes.

[0254] In certain embodiments, the composition may also contain one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers such as citric acid / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amounts needed to maintain the pH of the composition within an acceptable range. In other embodiments, the composition may also contain one or more salts in the amount needed to adjust the osmolality of the composition within an acceptable range. Such salts include salts having sodium, potassium, or ammonium cations with chloride, citric acid, ascorbic acid, boric acid, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0255] In some embodiments, sustained-release preparations may be used. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the engineered scaffold protein of the present disclosure, in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and y-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods. If encapsulated antibodies remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational stabilization strategies can be devised. For example, if the aggregation mechanism is found to be intermolecular S--S bond formation via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from an acidic solution, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions. In certain circumstances, pharmaceutical compositions can be delivered in a controlled-release system. In one embodiment, this may be a pump. In another embodiment, polymeric materials may be used. In yet another embodiment, the controlled-release system can be placed in close proximity to the target of the composition, thus requiring only a small fraction of the systemic dose.

[0256] In some embodiments, engineered scaffold protein can be administered with one or more drugs that can promote the engineered scaffold protein to penetrate the blood-brain barrier.In some embodiments, engineered scaffold protein can be linked with a viral vector, for example, to make the engineered scaffold protein more effective or increase the transport across the blood-brain barrier.For example, drug delivery can be carried out by administering an adenoviral vector to motor neurons in muscle tissue.Direct delivery of vector to the brain includes, but is not limited to, the striatum, thalamus, hippocampus, or substantia nigra.

[0257] In embodiments, engineered scaffold proteins can be linked or conjugated to agents that confer desirable pharmaceutical or pharmacodynamic properties. In some embodiments, engineered scaffold proteins can be coupled to substances that facilitate penetration of or transport across the blood-brain barrier, such as antibodies to the transferrin receptor. In some embodiments, osmotic blood-brain barrier disruption can be achieved by the use of sugars such as mesoerythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonisol, or the like. The composition can be supplemented by infusion of glucose-coated polymeric nanocarriers, such as glucose-coated lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine.

[0258] The compositions herein can be administered alone or in combination with another therapeutic agent, which may be administered prior to, simultaneously with, sequentially with, or following administration of the composition.

[0259] The compositions disclosed herein, including the engineered scaffold proteins described herein, can also contain two or more active agents, such as active agents with complementary activities that do not adversely affect each other, as needed for the particular indication being treated. For example, the compositions can further include an anti-inflammatory drug, a therapeutic protein, a steroid, an analgesic, a nonsteroidal anti-inflammatory drug, a corticosteroid, an immune system modulating drug, an additional engineered scaffold protein, two or more of the foregoing, or any combination thereof.

[0260] How to use Provided herein are compositions and methods for binding to target peptides, including the use of the engineered scaffold proteins described herein or fusion proteins thereof. Binding of a target peptide by the engineered scaffold proteins described herein or fusion proteins thereof can induce biological or therapeutic activity. For example, binding of a target peptide by the engineered scaffold proteins described herein or fusion proteins thereof can activate a binding site, inhibit or mask a binding site, induce conformational bias, prevent nuclear localization, disrupt membrane binding, modulate macromolecular interactions, mask or sequester post-translational modifications (PTMs), prevent proprotein processing, induce cell lysis, block substrate recruitment, or the like, or any combination thereof.

[0261] Also provided herein are compositions comprising an engineered scaffold protein or a fusion protein thereof described herein, wherein the engineered scaffold protein or a fusion protein thereof is bound, fused, conjugated, or otherwise attached to a therapeutic partner. Thus, in some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can deliver or localize the therapeutic partner to the target peptide, a cellular microenvironment containing the target peptide, a cell containing the target peptide, or the extracellular environment adjacent to or containing the target peptide. Thus, in some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce a biological or therapeutic activity provided by a therapeutic partner described herein. For example, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce cell death, induce proteolytic cleavage, modulate PTMs, induce dephosphorylation, induce protein isomerization, induce protein disaggregation and / or refolding, modulate ATPase and / or GTPase activity, induce an immune response, or the like, or any combination thereof.

[0262] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can activate the binding site. In some embodiments, activation of the binding site includes activation of a signaling pathway. In some embodiments, the signaling pathway that can be activated by an engineered scaffold protein or fusion protein thereof can include GPCR, ion channel, protein pump, enzyme-linked receptor, single-pass transmembrane protein, or secreted protein. Examples of signaling pathways that can be activated by the engineered scaffold proteins described herein include MAPK-PK, RAS / RAF, RHO, FAK1, MEK / MAPK, MAK, MKK, AKT, PI3K-AKT, EGF receptor, Her2 receptor, Her3 receptor, Her4 receptor, p38, NF-κB, NGF, NT-3, NT-4, BDNF, JNK, neurotrophins, PLC-γ1, estrogen receptor, progesterone receptor, androgen receptor. Signal transduction pathways include gen receptor, GPER30, PIK3 / PTEN, VEGF receptor pathway inhibitor, cell adhesion, TGF beta / SMAD, WNT, Hedgehog / GLI, HIF1 alpha, JAK / STAT, Notch, CD95 / Apo1L, mesolimbic dopamine, mesocortical dopamine, nigrostriatal dopamine, serotonergic pathway, acetylcholine, GABAergic, glutamatergic, Gl / S translocation regulation, DNA damage regulation, and apoptosis. In some embodiments, the signal transduction pathways include other signal transduction pathways disclosed herein. Thus, in some embodiments, provided herein are methods of activating signal transduction pathways, including the use of the engineered scaffolds described herein.

[0263] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can inhibit or mask the binding site. In some embodiments, inhibiting or masking the binding site comprises disrupting protein binding to one or more binding partners. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can form an intramolecular interaction with the target peptide. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can form an intermolecular interaction with the target peptide. In some embodiments, intramolecular and / or intermolecular interactions with the target peptide result in blocking of the protein comprising the target peptide. In some embodiments, intramolecular and / or intermolecular interactions can sterically mask the target protein binding site. In some embodiments, an engineered scaffold protein or fusion protein thereof can disrupt protein binding activity by blocking an active site, blocking a hydrophobic pocket, or causing steric hindrance on the surface of the target. In some embodiments, binding of a target peptide on a protein by an engineered scaffold protein or fusion protein thereof described herein can partially or fully block, mask, inhibit, or neutralize the biological activity of the protein. In some embodiments, binding of a target peptide on a protein by an engineered scaffold protein or fusion protein thereof described herein can reduce access to the protein's binding site. In some embodiments, binding of a target peptide on a protein by an engineered scaffold protein or fusion protein thereof described herein can block access to the protein's binding site. In some embodiments, binding of a target peptide on a protein by an engineered scaffold protein or fusion protein thereof described herein can inhibit signaling of the protein. In some embodiments, binding of a target peptide on a protein by an engineered scaffold protein or fusion protein thereof described herein can block the protein's signaling pathway.In some embodiments, the binding of a target peptide on a protein by the engineered scaffold protein or its fusion protein described herein can inhibit the binding of signaling molecules.Thus, in some embodiments, provided herein is a method for blocking the binding of a protein to one or more binding partners or inhibiting the biological activity of a protein, comprising using the engineered scaffold described herein.A schematic model of blocking by the engineered scaffold protein or its fusion protein described herein can be seen in Figure 33.

[0264] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce a conformational bias. In some embodiments, inducing a conformational bias involves stabilizing or locking a protein comprising the target peptide in a conformational state. In some embodiments, the conformational state is an active conformational state. In some embodiments, the conformational state is a partially active conformational state. In some embodiments, the conformational state is an inactive conformational state. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein selectively binds to a target peptide comprised in a protein, and the binding may not affect the function of the protein comprising the target peptide. Thus, in some embodiments, use of an engineered scaffold protein or fusion protein thereof described herein can stabilize a protein comprising a target peptide in a functional conformational state. In some embodiments, the functional conformational state is an active, basal, partially active, or inactive conformational state. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can correct a misfolded protein. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can correct a misfolded protein. In some embodiments, correcting a misfolded protein can restore the biological activity or function of the protein. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can prevent protein dissociation. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can prevent degradation of one or more proteins. Thus, in some embodiments, provided herein are methods for stabilizing or immobilizing proteins, including target peptides, comprising using an engineered scaffold described herein.

[0265] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can prevent nuclear localization. In some embodiments, preventing nuclear localization includes blocking nuclear transport of a protein comprising the target peptide. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can block a nuclear localization signal of the protein. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can block a nuclear export signal of the protein. In some embodiments, preventing nuclear localization by binding of a target peptide by an engineered protein described herein can occur in the presence of a nuclear localization signal. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can prevent one or more protein-protein interactions of the protein. In some embodiments, the protein-protein interaction can result in nuclear localization in the form of a complex. Thus, in some embodiments, binding of a target peptide by an engineered protein or fusion protein thereof described herein can prevent the protein from forming a complex and translocating to the nucleus. Thus, in some embodiments, provided herein are methods of preventing nuclear localization comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[0266] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can disrupt membrane binding. In some embodiments, disruption of membrane binding comprises reducing or preventing attachment or insertion of a protein comprising the target peptide into a cell membrane. In some embodiments, attachment can be prevented by blocking access to hydrophobic residues of the protein. In some embodiments, disruption of membrane binding can comprise an increase in soluble protein. In some embodiments, disruption of membrane binding can result in aberrant translocation of the protein to the cytoplasm and / or intracellular membranes of a cell in vitro. In some embodiments, disruption of membrane binding can result in aberrant translocation of the protein to the cytoplasm and / or intracellular membranes of a cell in vivo. In some embodiments, aberrant translocation of the protein can result in reduced access to a pathway. In some embodiments, reduced access to a pathway can prevent normal signaling of a membrane-bound partner molecule. Thus, in some embodiments, provided herein are methods of disrupting membrane binding comprising use of an engineered scaffold protein or fusion protein thereof described herein.

[0267] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can modulate a macromolecular interaction. In some embodiments, the macromolecular interaction includes multivalent binding, dynamic binding, and folding-binding interactions. In some embodiments, modulation of a macromolecular interaction can involve any suitable macromolecule. Non-limiting examples of macromolecules include disaccharides, polysaccharides, proteins, lipids, fatty acids, nucleic acids, cross-linked or non-cross-linked polymers, organometallic compounds, and other polymeric scaffolds. In some embodiments, the macromolecule comprises an aggregate of two or more macromolecules. In some embodiments, the macromolecular interaction includes protein-protein interactions, protein-DNA interactions, and protein-RNA interactions. In some embodiments, modulation of a macromolecule involves binding of a macromolecule to a protein. In some embodiments, the macromolecule bound to the protein can be a protein binding partner, antigen, epitope, peptide, ligand, receptor, carbohydrate, chemical, small molecule, or inhibitor. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof can result in increased protein binding to the macromolecule. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof can result in decreased protein binding to the macromolecule. In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof can be used to inhibit binding of one or more macromolecules to a protein. In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof increases binding, decreases binding, or changes the conditions under which a protein binds to a macromolecule. Thus, in some embodiments, provided herein are methods for modulating macromolecular interactions, comprising using the engineered scaffold protein or a fusion protein thereof described herein.

[0268] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can mask or sequester a post-translational modification (PTM). In some embodiments, masking or sequestering of a PTM can involve one or more PTMs. Examples of PTMs can include phosphorylation, acetylation, methylation, N-linked glycosylation with various sugars, O-linked glycosylation with various sugars, citrullination or deimination, crotonylation, butyrylation, ubiquitination, C-mannosylation, methionine oxidation, sulfation, amidation, sumolation, S-nitrosylation or nitrosylation, NEDDylation, deimination, OclcNAc, ADP-ribosylation, farnesylation, UFMylation, prenylation, myristoylation, S-palmitoylation, formylation, carboxylation, hydroxylation, and cis-trans isomerization of proline. In some embodiments, binding of a target peptide by an engineered scaffold protein or its fusion protein can be used to partially inhibit access to a PTM on a protein. In some embodiments, the protein may include, but is not limited to, a chaperone protein, an effector protein, an assembler protein, a scavenger protein, a degradation protein, or an enzyme. In some embodiments, binding of a target peptide by an engineered scaffold protein or its fusion protein can be used to inhibit access to a PTM on a protein. In some embodiments, binding of a target peptide by an engineered scaffold protein or its fusion protein can be used to block and / or sequester the PTM from an effector protein. In some embodiments, binding of a target peptide by an engineered scaffold protein or its fusion protein can be used to block the PTM from an enzyme that catalyzes the removal of the PTM. In some embodiments, blocking the PTM can reduce the functional state in which the protein can exist in a cell. In some embodiments, blocking the PTM can reduce the activity of the protein.Thus, in some embodiments, provided herein are methods of shielding or sequestering PTMs that involve the use of an engineered scaffold protein or fusion protein thereof described herein.

[0269] In some embodiments, binding of a target peptide by an engineered scaffold protein described herein, or a fusion protein thereof, can modulate a PTM. Thus, in some embodiments, modulating a PTM includes stimulating the activity of an endogenous or exogenous PTM enzyme that can generate and / or remove a PTM. In some embodiments, an endogenous or exogenous PTM enzyme can effect a PTM change. In some embodiments, the PTM change can be phosphorylation, acetylation, methylation, N-linked glycosylation with various sugars, O-linked glycosylation with various sugars, citrullination or deimination, crotonylation, butyrylation, ubiquitination, C-mannosylation, methionine oxidation, sulfation, amidation, sumolation, S-nitrosylation or nitrosylation, N-nitrosylation, deimination, OclcNAc, ADP-ribosylation, farnesylation, UFMylation, prenylation, myristoylation, S-palmitoylation, formylation, carboxylation, hydroxylation, or the like. , proline cis-trans isomerization, deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, de-NEDDylation, removal of OclcNAc, de-ADP-ribosylation, demethylation, dehydroxylation, defarnesylation, de-UFMylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof.Examples of endogenous or exogenous PTM enzymes include carboxylate-amine ligases, cyclases, dehydrogenases, cyclodehydratases, decarboxylases, epimerases, hydroxylases, peptidases, dehydratases, transferases, esterases, oxygenases and isomerases, ubiquitin ligases, SUMO transferases, methyltransferases, demethylases, acetyltransferases, glycosyltransferases, palmitoyltransferases and / or related hydrolases, lanthionine bond-forming enzymes, cytolysin-forming enzymes, cyanobactin-forming enzymes, thiopeptide-forming enzymes, conopeptide-forming enzymes, microviridin-forming enzymes, cyclotide-forming enzymes, bacteriocin-forming enzymes, and subtilosin-forming enzymes. In some embodiments, conjugation of a target peptide by an engineered scaffold protein or its fusion protein increases the biological activity of the endogenous or exogenous PTM enzyme. In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof reduced the biological activity of an endogenous or exogenous PTM enzyme. Thus, in some embodiments, provided herein are methods for modulating PTMs, comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[0270] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce dephosphorylation. In some embodiments, inducing dephosphorylation comprises increasing the enzymatic activity of a phosphatase enzyme. In some embodiments, the engineered scaffold protein or fusion protein thereof increases the binding of the phosphatase to a target protein, including the target peptide. Examples of phosphatase enzymes include, but are not limited to, bacterial alkaline phosphatase, mammalian alkaline phosphatase, plant acid phosphatase, mammalian acid phosphatase, and alkaline phosphatase conjugates. Thus, in some embodiments, provided herein are methods for inducing dephosphorylation, comprising the use of an engineered scaffold protein or fusion protein thereof described herein.

[0271] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce protein isomerization. In some embodiments, the induction of protein isomerization involves activation of an isomerase enzyme. In some embodiments, activation of the isomerase enzyme results in increased racemization of the target protein. In some embodiments, activation of the isomerase enzyme results in increased isomerization of the target protein. In some embodiments, the isomerase enzyme can be an aldose triosephosphate isomerase, a bisphosphoglycerate mutase, a triosephosphate isomerase, a phosphomutase, an epimerase, a racemase, and a carbon skeleton mutase, or a photoisomerase. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can induce isomerization of an amino acid residue. In some embodiments, the amino acid residue is proline. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can increase the isomerization of proline. In some embodiments, the isomerization of proline is catalyzed by proline isomerase. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein increases the biological activity of proline isomerase, resulting in one or more conformations of proline. In some embodiments, the one or more conformations of proline include a cis conformation and a trans conformation. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein increases cis-trans isomerization of proline. Thus, in some embodiments, provided herein are methods for inducing protein isomerization, comprising the use of an engineered scaffold protein or fusion protein thereof described herein.

[0272] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can modulate ATPase / GTPase activity. In some embodiments, modulating ATPase / GTPase activity comprises activating the ATPase / GTPase. In some embodiments, modulating ATPase / GTPase activity comprises inhibiting the ATPase / GTPase. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein increases ATP hydrolysis. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein increases GTP hydrolysis. In some embodiments, modulating ATPase activity can be used to treat a disease. In some embodiments, the disease comprises aberrant ATPase activity. In some embodiments, modulating GTPase activity can be used to treat a disease. In some embodiments, the disease comprises aberrant GTPase activity.Examples of diseases associated with abnormal ATPase / GTPase activity include, but are not limited to, stroke, cardiovascular disease (e.g., angina pectoris, myocardial infarction, chronic ischemic heart disease, hypertensive heart disease, pulmonary heart disease, valvular heart disease, rheumatic fever, rheumatic heart disease, endocarditis, mitral valve prolapse, aortic valve stenosis, congenital heart disease, valvular and vascular obstructive lesions, atrial or ventricular septal defects, patent ductus arteriosus, and myocardial disease. ), Wilson's disease, Menkes syndrome, renal disorders (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes mellitus, neoplasms, sickle cell disease, chronic inflammatory diseases, acute tubular necrosis, acute renal failure, polycystic kidney disease, medullary sponge kidney, medullary cystic kidney disease, renal diabetes, renal tubular acidosis, renal cytotoxicity, pulmonary disorders (e.g., emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathic pulmonary hematosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonia, chronic pulmonary arterial disease, acute and rapidly progressive renal failure, chronic renal failure, and nephrolithiasis), hypotension, hypertension, ischemic disorders, neurological disorders (e.g., Alzheimer's disease and Parkinson's disease), muscle disorders, pulmonary disorders (e.g., emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathic pulmonary hematosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonia, chronic These include disorders such as idiopathic interstitial pneumonia, fibrosing alveolitis, Hermann-Rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis, Wegener's granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), liver disorders (e.g., hepatic vein thrombosis, portal vein obstruction, thrombosis, hepatitis, and cirrhosis), proliferative disorders (e.g., neoplasms or tumors, such as carcinomas, sarcomas, adenomas, and myeloid leukemias), and disorders in which a positive inotropic effect is desired. Accordingly, in some embodiments, provided herein are methods for modulating ATPase / GTPase activity, comprising the use of the engineered scaffold proteins described herein or fusion proteins thereof.

[0273] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can prevent proprotein processing. In some embodiments, preventing proprotein processing comprises preventing proteolytic cleavage. In some embodiments, preventing proteolytic cleavage involves binding to, partially binding to, blocking, or burying a catalytic site from one or more proteases. In some embodiments, the one or more proteases may include proprotein convertases, serine proteases, metalloproteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases. In some embodiments, preventing proprotein processing may result in a reduction in the number of bioactive peptides. In some embodiments, preventing proprotein processing may result in a reduction in the number of bioactive proteins. In some embodiments, preventing proprotein processing may result in a reduction in the number of active enzymes. Thus, in some embodiments, provided herein are methods of preventing proprotein processing, comprising the use of an engineered scaffold protein or fusion protein thereof described herein.

[0274] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce protein disaggregation and / or refolding. In some embodiments, the induction of protein disaggregation and / or refolding is accompanied by activation of a chaperone enzyme. Examples of chaperone enzymes include Hsp90 family proteins, Hsp70 family proteins, immunophilin family proteins, peptidase C56 family proteins, Pplase family proteins, 14-3-3 family proteins, small heat shock protein family proteins, small GTPase family proteins, Hsp40 (DnaJ) family proteins, and any of clusterin, Grp170, calreticulin, Hsp105, Hsp70-interacting protein (CHIP), alpha-crystallin, or a combination thereof. In some aspects, activation of a chaperone enzyme results in an increase in the number of refolded proteins. In some aspects, activation of a chaperone enzyme results in an increase in the number of disaggregated proteins. In some embodiments, binding of a target peptide by the engineered scaffold protein described herein or a fusion protein thereof can increase the refolding of a multimeric protein. In some embodiments, the multimeric protein can include, but is not limited to, a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or nonamer. The multimeric protein subunits can be identical (such as homodimers), or one or more can be different within the native protein (such as heterodimers). Thus, in some embodiments, provided herein are methods for inducing protein disaggregation and / or refolding, comprising the use of the engineered scaffold protein described herein or a fusion protein thereof.

[0275] In some embodiments, binding of a target peptide by an engineered scaffold protein described herein or a fusion protein thereof can induce proteolytic cleavage. In some embodiments, inducing proteolytic cleavage comprises increasing proteolytic cleavage. In some embodiments, increasing proteolytic cleavage comprises increasing the accessibility of a catalytic enzyme to the protein. In some embodiments, the catalytic enzyme is a protease. In some embodiments, increasing proteolytic cleavage involves binding, partially binding, stabilizing, or exposing a catalytic site in the protein to one or more proteases. In some embodiments, the one or more proteases may include proprotein convertases, serine proteases, metalloproteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases. In some embodiments, inducing proteolytic cleavage can result in an increase in the number of bioactive peptides. In some embodiments, inducing proteolytic cleavage can result in an increase in the number of bioactive proteins. In some embodiments, inducing proteolytic cleavage can result in an increase in the number of active enzymes. Thus, in some embodiments, provided herein are methods of inducing proteolytic cleavage comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[0276] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce cell lysis. In some embodiments, inducing cell lysis can induce cell death. In some embodiments, inducing cell lysis and / or cell death comprises activating an apoptotic pathway, an autophagic cell death pathway, a necrotic pathway, complement-dependent cytotoxicity, or antibody-dependent cellular cytotoxicity. In some embodiments, a therapeutic method disclosed herein can comprise a pharmacological drug conjugated to an engineered scaffold protein or fusion protein thereof and delivered to a specific cell as a therapeutic agent. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can be used in therapeutic methods where induction of apoptosis is therapeutically desirable. In some embodiments, the therapeutic method comprises increasing apoptosis. In some embodiments, increased apoptosis can be used to selectively kill desired target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce more apoptosis in desired target cells than non-target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can be used in therapeutic methods where induction of autophagy is therapeutically desirable. In some embodiments, the therapeutic method comprises increasing autophagy. In some embodiments, increasing autophagy can be used to selectively kill desired target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce more autophagy in desired target cells than in non-target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can be used in therapeutic methods where induction of necrosis is therapeutically desirable. In some embodiments, the therapeutic method comprises increasing necrosis. In some embodiments, increasing necrosis can be used to selectively kill desired target cells.In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce greater necrosis in desired target cells than in non-target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can be used in therapeutic methods where induction of complement-dependent cytotoxicity is therapeutically desirable. In some embodiments, the therapeutic methods include increased complement-dependent cytotoxicity. In some embodiments, increased complement-dependent cytotoxicity can be used to selectively kill desired target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce greater complement-dependent cytotoxicity in desired target cells than in non-target cells. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can be used in therapeutic methods where induction of antibody-dependent cellular cytotoxicity is therapeutically desirable. In some embodiments, the therapeutic methods include increased antibody-dependent cellular cytotoxicity. In some embodiments, increased antibody-dependent cellular cytotoxicity can be used to selectively kill desired target cells. In some embodiments, binding of a targeting peptide by an engineered scaffold protein or fusion protein thereof described herein can induce greater antibody-dependent cellular cytotoxicity in desired target cells than in non-target cells. In some embodiments, the therapeutic methods described herein can be used to treat diseases. In some embodiments, diseases include, but are not limited to, cancer, infectious diseases, neurodegenerative diseases, cardiovascular diseases, pulmonary diseases, muscular diseases, liver diseases, autoimmune and / or inflammatory diseases, and proteinopathies. In some embodiments, the therapeutic methods described herein can be used to selectively kill cancer cells that are resistant to other therapies. Thus, in some embodiments, provided herein are methods of inducing cell lysis, comprising the use of an engineered scaffold protein or fusion protein thereof described herein.

[0277] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can block substrate recruitment. In some embodiments, blocking substrate recruitment inhibits protein-protein interactions. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein prevents protein-substrate binding. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can limit access to the active site. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can block an essential substrate recruitment site. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can block a catalytic site. In some embodiments, an engineered scaffold protein or fusion protein thereof can physically block substrate binding. Thus, in some embodiments, provided herein are methods of blocking substrate recruitment comprising the use of an engineered scaffold protein or fusion protein thereof described herein.

[0278] In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can induce an immune response. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can stimulate an immune response in vivo. In some embodiments, stimulating an immune response results in upregulation of genes encoding cytokines, chemokines, or lymphokines. Examples of such genes include alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFP, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL-15. In some embodiments, binding of a target peptide by an engineered scaffold protein or fusion protein thereof described herein can be used in the form of a vaccine. In some embodiments, the vaccine comprises an inactivated vaccine, a live attenuated vaccine, a messenger RNA (mRNA) vaccine, a subunit, recombinant, polysaccharide, and conjugate vaccine, a neoepitope vaccine, a neurodegenerative vaccine, an infectious disease vaccine, a toxoid vaccine, or a viral vector vaccine. Thus, the vaccine may be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means for delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, a live attenuated vaccine, or an inactivated vaccine. Thus, in some embodiments, provided herein are methods for inducing an immune response, comprising the use of an engineered scaffold protein described herein or a fusion protein thereof.

[0279] Therapeutic Use Therefore, the engineered scaffold proteins or fusion proteins thereof disclosed herein can be developed as therapeutic molecules. In some embodiments, the therapeutic molecule can bind to any protein on the cell surface, a secreted protein in the extracellular space, or a protein in an intracellular compartment. The protein target of the therapeutic molecule can be a receptor, a cell surface marker, a membrane-bound protein, an enzyme, an intracellular protein, a signaling pathway component, or other protein. The therapeutic molecule can act as an inhibitor that binds to the target protein and inhibits important biological activity. Therapeutic molecules can also act to regulate protein-protein interactions, either as antagonists or agonists. Therapeutic molecules may also regulate the enzymatic activity of the target protein or another protein that interacts with the target protein. For example, by fusing a protease enzyme to the engineered scaffold, the therapeutic molecule can be engineered to specifically bind to and cleave the target protein. In some embodiments, the modality prevents protein aggregation by binding to the target protein and thereby altering its biophysical properties. As an example, alpha-synuclein is a precursor to plaque formation in many neurodegenerative diseases. Plaques form due to the aggregation of α-synuclein. By binding to the target protein and preventing its aggregation, it may be possible to treat neurodegenerative diseases. Similar to antibody-drug conjugates, pharmacological drugs can be conjugated to engineered scaffolds or their fusion proteins and delivered to specific cells as therapeutic agents.

[0280] Also provided herein are compositions and methods comprising an engineered scaffold protein or fusion protein thereof described herein and a therapeutic partner. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can be used to deliver the therapeutic partner to a target. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can function as a vehicle for delivering the therapeutic partner to a target. In some embodiments, the therapeutic partner is a therapeutic peptide. In some embodiments, the therapeutic peptide can be loaded onto an engineered scaffold protein or fusion protein thereof described herein. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can form a covalent bond with the therapeutic peptide. In some embodiments, the covalent bond is a disulfide bond between a cysteine ​​residue on an engineered scaffold protein or fusion protein thereof described herein and a cysteine ​​residue on the therapeutic peptide. In some embodiments, the therapeutic peptide can be loaded onto a portion of the amino acid chain of an engineered scaffold protein or fusion protein thereof described herein. In some embodiments, the amino acid chain of an engineered scaffold protein or fusion protein thereof described herein may include an N-terminal peptide, a C-terminal peptide, or a peptide protruding from a mid-loop on an engineered scaffold protein or fusion protein thereof described herein. In some embodiments, an engineered scaffold protein or fusion protein thereof may be linked to a therapeutic peptide at the N-terminus of the engineered scaffold protein or fusion protein thereof described herein. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein may be linked to a therapeutic peptide by an N-terminal linker. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein may be linked to a therapeutic peptide at the C-terminus of the engineered scaffold protein or fusion protein thereof.In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can be linked to a therapeutic peptide via a C-terminal linker. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can be linked to a therapeutic peptide at any residue on the engineered scaffold protein or fusion protein. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can form a non-covalent bond with a peptide drug. In some embodiments, the non-covalent bond can include, but is not limited to, van der Waals forces, salt bridges, or hydrogen bonds. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein and the peptide drug are linked by a linker. In some embodiments, the linker is a proteolytically cleavable linker. Thus, the engineered scaffold proteins or fusion proteins thereof can function as a vehicle for a therapeutic peptide.

[0281] Binding of an engineered scaffold protein or fusion protein thereof described herein to a therapeutic peptide can serve multiple functions, including, but not limited to, cell specificity, tissue specificity, and half-life extension. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can be engineered to bind to a specific target. In some embodiments, the specific target is overexpressed in a desired cell type. In some embodiments, the desired cell type is a diseased cell. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can selectively bind to diseased cells over normal cells. Thus, in some embodiments, an engineered scaffold protein or fusion protein thereof described herein can specifically deliver a therapeutic peptide to a desired cell type. In some embodiments, a therapeutic peptide can be embedded at the interface of an engineered scaffold protein or fusion protein thereof described herein. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can extend the half-life of a therapeutic peptide. In some embodiments, an engineered scaffold protein or fusion protein thereof described herein can increase the stability of a therapeutic peptide. In some embodiments, an engineered scaffold protein or fusion protein thereof can protect a therapeutic peptide from proteolytic enzymes. Thus, in some embodiments, engineered scaffold proteins or fusion proteins thereof can be used to increase the target specificity and half-life of therapeutic peptides.

[0282] Conjugation of the engineered scaffold proteins or fusion proteins thereof described herein to a therapeutic peptide can be used for targeted release of the therapeutic peptide. In some embodiments, targeted release of the therapeutic peptide can include endocytosis, linker cleavage, release from the binding interface, uncontrolled release, etc. In some embodiments, the therapeutic peptide can be released from the engineered scaffold proteins or fusion proteins thereof described herein during the endocytosis process. In some embodiments, the therapeutic peptide can be released from the engineered scaffold proteins or fusion proteins thereof described herein by linker cleavage of specific linkers under one or more cellular environmental conditions. In some embodiments, the specific linker can include a protease-sensitive peptide linker, an acid-sensitive hydrazone linker, a pH-sensitive linker, or a glutathione-sensitive disulfide linker. In some embodiments, binding of the engineered scaffold proteins or fusion proteins thereof to a target cell can induce a conformational change that releases the therapeutic peptide from the interface of the engineered scaffold proteins or fusion proteins thereof described herein. In some embodiments, the therapeutic peptide can bind to a therapeutic target while attached to the engineered scaffold proteins or fusion proteins thereof described herein via a long linker. In some embodiments, the long linker can include a long peptide linker or a long non-peptide linker, hi some embodiments, the long peptide linker has a length of 10-50 amino acids.In some embodiments, the long peptide linker has 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids, or 50 amino acids. Thus, the engineered scaffold proteins or fusion proteins thereof described herein can be used as a targeted drug delivery system for therapeutic peptides.

[0283] Also provided herein are methods of treatment comprising the engineered scaffold proteins or fusion proteins thereof described herein. In some embodiments, the engineered scaffold proteins or fusion proteins thereof can enhance therapeutic treatment of a disease in a subject. In some embodiments, therapeutic treatments can include chimeric antigen receptor (CAR) T-cell therapy and lysosome-targeted chimeric (LYTAC) therapy. CAR-T cell therapy is a type of cancer immunotherapy treatment that uses immune cells called T cells that have been genetically modified to more effectively locate and destroy cancer cells. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can replace the binding domain of a chimeric antigen receptor (CAR). In some embodiments, the binding domain of a CAR is an scFV domain. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can be hinge-linked to the endogenous transmembrane domain of a CAR. In some embodiments, the engineered scaffold proteins or fusion proteins thereof described herein can be linked to the endogenous transmembrane domain of a CAR. In some embodiments, the engineered scaffold protein or fusion protein thereof described herein linked to a CAR can improve the stability, half-life, and specificity of the CAR. Therefore, the engineered scaffold protein or fusion protein thereof can be used to enhance CAR T-cell therapy. Lysosomal targeting chimeras (LYTACs) are known in the art to induce degradation of extracellular and membrane proteins via the endosomal-lysosomal pathway. In some embodiments, the engineered scaffold protein or fusion protein described herein can replace antibodies fused to a ligand. In some embodiments, the ligand can bind to a receptor. In some embodiments, the receptor can mediate cellular internalization. In some embodiments, a ligand can be conjugated to the engineered scaffold protein or fusion protein described herein for use as a LYTAC to degrade targets on the cell membrane.Thus, the engineered scaffold proteins described herein linked to LYTACs, or fusion proteins thereof, can improve the stability, half-life, and specificity of LYTACs.

[0284] Therefore, the present specification discloses a method for preventing / treating a disease or disorder associated with a target peptide.The present specification also discloses a method for treatment, which comprises administering an effective amount of an engineered scaffold protein, a fusion protein thereof, or a pharmaceutical composition comprising the same to a subject in need thereof.The subject in need thereof may be a subject suffering from or at risk of developing a disease or disorder associated with a target peptide.

[0285] Devices and diagnostic agents Provided herein are compositions and methods for detecting the presence or absence of a biological analyte, including the use of a detection agent comprising the engineered scaffold protein described herein or a fusion protein thereof. The biological analyte described herein refers to peptides, small molecules / metabolites (e.g., dNTPs), nucleic acids (e.g., double-stranded DNA, single-stranded DNA, RNA, circular DNA, circular RNA, amino acids, etc.), or whole cells, cell compartments / organelles, antibodies, serum components, hemoglobin, etc.

[0286] Detecting the presence or absence of a biological analyte using a detection agent comprising an engineered scaffold protein or a fusion protein thereof described herein involves binding to and labeling the biological analyte. For example, labeling a biological analyte with an engineered scaffold protein or a fusion protein thereof described herein includes conjugating a specific tag to the engineered scaffold protein or a fusion protein thereof described herein. Examples of specific tags include imaging agents, polypeptides linked to imaging agents, chelators, fluorescent labels, luciferase enzymes, self-labeling proteins, peptide tags (e.g., ALFA tag, AviTag, FLAG tag, HA tag, iCapTag, etc.), and biotin carboxyl carrier proteins. By labeling the engineered scaffold protein or a fusion protein thereof with a specific tag, the presence or absence of a biological analyte in a sample can be determined by the interaction of the engineered scaffold protein or a fusion protein thereof with the biological analyte. Such interaction allows the identification of the specific biological analyte that interacts with the engineered scaffold or fusion protein. Similarly, engineered scaffolds or fusion proteins may be engineered against specific peptide sequences encoding post-translational modifications (PTMs) to identify peptide / protein sequences encoding PTMs. Detection can be for protein sequencing, protein fingerprinting, protein quantification, or protein identification. In some embodiments, detection agents comprising the engineered scaffold proteins described herein or fusion proteins thereof can be used to detect biological analytes or biomolecules. In some embodiments, biological analytes or biomolecules include, but are not limited to, small molecules / metabolites (e.g., dNTPs), nucleic acids (e.g., double-stranded DNA, single-stranded DNA, RNA, circular DNA, circular RNA, amino acids, etc.), glucose, potassium, lactate, uric acid, sodium, chloride, proteins (e.g., receptors, channels, soluble proteins, etc.), lipids, and cells.One example of detecting such biological analytes with any of the engineered scaffold proteins disclosed herein is to generate a diverse set of engineered scaffold proteins that are coupled to an orthogonal set of peptide sequences that serve as peptide barcodes.

[0287] In some embodiments, a detection agent comprising an engineered scaffold protein or fusion protein thereof described herein is provided in the form of a device. In some embodiments, the device comprises a solid support and a detection agent holder. In some embodiments, the detection agent holder comprises a site on the device to which a liquid sample is added. Examples of liquid samples include urine, blood, sweat, mucus, interstitial fluid, chyme, saliva, bile, semen, cerebrospinal fluid, sputum, pus, breast milk, and synovial fluid. By adding a liquid sample containing a biological analyte and / or a biological molecule to the device via the detection agent holder of the device, the detection agent comprising an engineered scaffold protein or fusion protein thereof described herein can bind to the biological analyte and / or biological molecule to be measured or quantified. In some embodiments, the device may comprise a solid support such as a porous membrane or a microfluidic chip. In some embodiments, the device is a microfluidic chip. Microfluidic chips can be fabricated by methods known in the art, for example, by preparing flow channels having mixing and reaction zones, one or more inlets, and a waste reservoir in a small piece of glass or plastic. The inlet is used to inject a liquid sample containing the substance to be measured, and separate inlets for injecting wash solutions and / or enzyme substrate solutions into the channel may be provided as needed. In some embodiments, the device is formed by a microfluidic chip comprising a device having only a biological analyte and / or biomolecule capture portion. In some embodiments, the biological analyte and / or biomolecule capture portion comprises an engineered scaffold protein or a fusion protein thereof described herein. In some embodiments, the device may be a kit comprising a detection agent comprising an engineered scaffold protein or a fusion protein thereof described herein.

[0288] In some embodiments, compositions and methods for detecting the presence or absence of a biological analyte include an assay system, wherein the kit includes a detection agent comprising an engineered scaffold protein or fusion protein described herein and a measurement device capable of measuring a signal generated from a reporter substance of the detection agent. In some embodiments, the detection agent is held in a detection agent holder. In some embodiments, the detection agent holder is combined with a measurement device. In some embodiments, the signal generated can include fluorescence, luminescence, color, turbidity, radiation, or a spectrometer signal. In some embodiments, spectrometers include, but are not limited to, optical absorption spectrometers, luminescence spectrometers, electron spectroscopy, mass spectrometers, time-of-flight spectrometers, and magnetic spectrometers. In some embodiments, the measurement device can include a liquid pump. In some embodiments, the liquid supply pump can be used to control the speed at which a liquid sample containing the biological analyte and / or biomolecules moves through the device.

[0289] In some embodiments, the device containing the detection agent comprises an engineered scaffold protein or a fusion protein thereof, and the engineered scaffold protein or a fusion protein thereof is immobilized on a solid support in a manner that preserves the three-dimensional structure of the engineered scaffold protein or fusion protein. In some embodiments, a liquid sample is added to the device containing the immobilized engineered scaffold protein or a fusion protein thereof. In some embodiments, the immobilized engineered scaffold protein or a fusion protein thereof forms a complex with a biological analyte or a biological molecule in the liquid sample, and a signal is generated upon binding. In some embodiments, the generated signal can be used to generate a standard treatment or a dose-effect curve. In some embodiments, the signal intensity measured when a liquid sample contains an unknown concentration of a biological analyte or a biological molecule can be inserted into a standard curve to determine the amount of the biological analyte or biological molecule. As used herein, a biological molecule refers to a protein (e.g., a receptor, a channel, a soluble protein, a membrane-bound protein, a post-translationally modified protein), a lipid, a carbohydrate, etc.

[0290] In some embodiments, described herein are assay methods for detecting, monitoring, or measuring a given ligand, such as a target ligand, in a sample, comprising inducing an expression vector described herein to express an engineered scaffold protein or a fusion protein thereof, then contacting the engineered scaffold protein or a fusion protein thereof with the sample, and observing whether the engineered scaffold protein or a fusion protein thereof interacts with the target ligand. In such embodiments, the method comprises the use of a polypeptide display system.

[0291] Further details and examples can be found in the Examples provided herein. Sequences and Tables [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 1-62

Table 1-63

Table 1-64

Table 1-65

Table 1-66

Table 1-67

Table 1-68

Table 1-69

Table 1-70

Table 1-71

Table 1-72

Table 1-73

Table 1-74

Table 1-75

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0292] The table below provides information about peptides known to bind to each human protein scaffold (i.e., "bound peptides"). [Table 3-1]

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

[0293] The following table shows exemplary sequences for use in binding units of the binding domains of the engineered scaffold proteins described herein. [Table 4]

[0294] The following table shows exemplary sequences for use in the hinge unit of the binding domains of the engineered scaffold proteins described herein. [Table 5]

[0295] The following table shows exemplary sequences contained in the disordered regions described herein. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10]

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

[0296] The following table shows exemplary sequences contained in the disordered regions described herein. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0297] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

[0298] Example 1. Detection of biological analytes using engineered scaffold proteins Engineered scaffold proteins generated from the scaffolds disclosed herein can target specific proteins present based on information from only one type of cell, allowing quantitative measures of cell number and relative ratios for different cell types to be determined. For example, by identifying a population of T cells and their relative numbers compared to B cells, engineered scaffolds can be designed for CD28 (CTLA), which is present in T cells but not B cells, and CD22, which is present in B cells but not T cells. Each engineered scaffold is labeled with a different fluorophore or peptide tag that binds to a fluorescent antibody. The labeled binding agent is incubated with the T cell and B cell populations, followed by washing to remove free binding agent. Flow cytometry is then used to sort and separate the cells by the two different colors. Quantitative assessment of the different cell types can then be performed.

[0299] Similar assays can be set up to examine the presence of other biological analytes using fluorescently labeled engineered scaffolds or engineered scaffolds with peptide tags that can bind to fluorescent antibodies. For example, engineered scaffolds can be designed for specific target proteins expressed by mRNAs that may be cellular biomarkers, allowing the presence of specific cells in a population of cells to be determined. By combining the assay with single-molecule microscopy, this assay can be made quantitative.

[0300] Example 2. Scaffold manipulation The following non-limiting examples demonstrate the development of engineered scaffolds that are designed to target specific epitopes.

[0301] C-myc is a well-validated cancer target, but its inherently disordered nature makes it extremely difficult to treat with drugs. To target this disordered region, we identified the sequence EPLVLHE, which is present in the disordered region of C-myc, as a target epitope.

[0302] A catalytically inactive TEV protease (C151A) is selected as a scaffold for binder development. A library of variants maintaining the C151A mutation (theoretical maximum of 10) is created. 8 (up to 1000) are generated using Twist Bioscience's SOLD approach, which distributes variants across the entire length of the enzyme sequence.

[0303] The library of variants is cloned into a yeast vector, fused to the Aga1 protein at either the N- or C-terminus, and further fused to a 6x histidine tag at the C-terminus of the Aga1-TEV variant fusion, and transfected into yeast cells.

[0304] Functionally expressed and displayed variants are incubated with magnetic beads bearing the recognition peptide EPLVLHE and selected for binding. Binders from this selection are washed, labeled with a tag-containing protein, and subjected to FACS sorting to separate binders from non-binders.

[0305] Based on the initial selection round, positive and negative binders are separated, and the yeast cells are subjected to NGS sequencing to identify binders and non-binders. Binders from the initial selection round are amplified and subjected to further screening rounds of more stringent binding conditions to obtain strong binders for the target sequence.

[0306] The data generated from each selection round is used to create a database of engineered scaffold proteins and their relative intensities against their targets. A flowchart demonstrating the scaffold engineering process can be seen in Figure 11.

[0307] Example 3. Binder development pipeline A non-limiting example of a binder development pipeline is shown in Figure 13. Yeast display of cell surface protein scaffolds is performed using well-known methods, such as those described in Angelini et al. (2015), Methods in Molecular Biology, "Protein Engineering and Selection Using Yeast Surface Display." The procedure begins with the design of a library of engineered scaffolds for each scaffold protein. A yeast strain in which the Aga1 gene is stably integrated into the chromosome is selected. The nucleic acid encoding the engineered scaffold protein fused to the Aga2 gene is cloned into a circular yeast display vector using Gibson assembly and further established protocols. The yeast library is transformed into competent cells and selected using the URA3 complementation method. The yeast library cells are first grown in YPD medium and then used to seed SD-CAA medium. The cells are passaged once, induced, and then the induced cells are used to screen for binders. Screening is performed in two steps using magnetic bead-based screening followed by flow cytometry.

[0308] In the first step, negative selection of magnetic bead-binding agents is performed by incubating the library with magnetic beads without immobilized targets to deplete the library of streptavidin-coated magnetic bead-binding agents. The flow-through from the negative selection is then incubated with magnetic beads with targets immobilized via biotin-streptavidin linkage. After stringent washing to remove non-binders, the beads are recovered and the binder library is isolated.

[0309] Yeast cells selected from the magnetic bead screening step are further subjected to fluorescence-activated cell sorting (FACS) to enrich for binders with higher affinity for the target protein. The yeast display vector is designed to have a tag epitope (His or FLAG) at the C-terminus of the engineered scaffold protein, allowing it to be labeled with an antibody. The target protein is tagged with a different tag (His, FLAG, or myc). Using two-color FACS sorting, only yeast cells that exhibit engineered scaffold proteins that bind to the target protein exhibit two colors and are collected. Repeated selection and screening cycles yield strong binders for the target protein.

[0310] Example 4. Monomeric MHC design The following non-limiting examples show various engineered monomeric MHC constructs and designs for engineered scaffold proteins derived from MHC proteins. The engineered scaffold proteins can be produced according to any of the methods and examples disclosed herein. [Table 8-1] [Table 8-2] [Table 8-3]

[0311] The specific linker sequences used in the MHC constructs are also listed below. [Table 9]

[0312] The (GGGGS)n linker is a flexible linker that is thought to increase solubility and stability. The LE linker is thought to increase the PK of the antibody (e.g., acts as a dipeptide linker). QSEAGSH (SEQ ID NO: 976) is an MHC I linker derived from the crystal structure of 2XPG. It is a linker between the alpha helix and the second part of the beta sheet.

[0313] Specific fusion partner sequences used in the MHC constructs disclosed herein are also listed below. [Table 10]

[0314] Chimera 1-4 constructs The constructs of each of Chimeras 1 to 4 listed in Table 8 are described as follows.

[0315] Chimera 1. The construct of Chimera 1 is shown below, where domains 1 and 3 can be part of the MHC II α or MHC II β chain, and domain 2 can be part of the MHC I α chain. [ka]

[0316] Chimera 2. The construct for Chimera 2 is shown below, where Domain 1 can be part of the MHC II α or β chain, and Domain 2 can similarly be part of the MHC II α or β chain. The two domains are connected by a short peptide linker. [ka]

[0317] Chimera 3. The construct of Chimera 3 is shown below, where domain 1 or domain 3 can be part of the MHC Iα chain, domains 2 and 4 can be part of the MHC IIα / β chain, and domains 2 and 3 are connected by a short peptide linker. [ka]

[0318] Chimera 4. The construct for Chimera 4 is shown below, where domain 1 can be part of the MHC Iα chain or the MHC IIα / β chain, and similarly for domain 2. [ka]

[0319] All of the above constructs may have C- or N-terminal fusions that may include a purification tag such as a His tag (HHHHHH), a FLAG tag, a Myc tag, a GST tag, a solubility tag such as SUMO, a protease cleavage tag such as a TEV protease cleavage tag (ENLYFS) or a thrombin cleavage tag, a serum albumin binding domain or a transferrin domain or a transferrin binding domain. (0001) Example 5. Generation of engineered scaffold proteins from inclusion bodies This example describes a method for producing the engineered scaffold proteins described herein. E. coli pET28 or pD451 vectors encoding the engineered scaffold proteins of interest were transformed into BL21 DE3 cells. Overnight starter cultures were grown from single colonies and then used to inoculate 1 L cultures containing the appropriate antibiotics and incubated at 37°C. Once the optical density reached 0.6-0.8, protein production was induced by the addition of 1 mM IPTG, and the flasks were maintained at 37°C with shaking for 16 hours. Cells were then harvested by centrifugation at 8000 x g for 10 minutes, resuspended in lysis buffer (50 mM Tris pH 8, 500 mM NaCl, 10 mM imidazole), and lysed using a sonicator. While maintaining the cells in an ice bath, the sonicator was set to pulse mode with a 15-second on time and a 45-second off time for a total of 10 cycles.

[0320] The cell lysate was centrifuged at 18,000 x g for 30 minutes to clarify the solution, and the cell pellet containing the target protein in inclusion bodies was saved, and the supernatant was discarded. The pellet was resuspended in 10 mL of urea buffer (50 mM Tris pH 8, 500 mM NaCl, 10 mM imidazole, 6 M urea) and incubated overnight at room temperature on a rotator to resolubilize the target protein from the inclusion bodies. The resolubilized solution was again clarified and centrifuged at 18,000 x g for 30 minutes to remove aggregates, and the supernatant was saved. The resolubilized protein was applied to a nickel column pre-equilibrated with lysis buffer and allowed to bind under gravity flow. The column was then thoroughly washed with wash buffer (50 mM Tris pH 8, 500 mM NaCl, 25 mM imidazole) to remove urea and refold the protein on the column. The target protein was eluted with elution buffer (50 mM Tris pH 8, 500 mM NaCl, 250 mM imidazole). The eluted protein was buffer exchanged into PBS, flash frozen, and stored.

[0321] Engineered scaffold proteins produced by the above methods include S001, S005-S007, S031, S043-S044, S049, S055-S060, S065-S069, S077-S078, S082, S090, S095-S096, S098-S102, S107, S117-S118, S132-S134, S139-S140, S143-S145, S161-S167, S169-S180, S183-S215, S227, S232, S236, S240, S242, S249, S258, and SEQ ID NO: 914.

[0322] Example 6: Characterization of engineered scaffold proteins This example characterizes the engineered scaffold proteins produced as described herein. The molecular weights of the engineered scaffold proteins produced from the method of Example 5 range from about 20 kDa to about 50 kDa. SDS PAGE was performed on the engineered scaffold protein (S177 (SEQ ID NO: 827)), which confirmed the expected size (21.92 kDa) on the gel, as demonstrated in Figure 15A, and can be produced in a soluble form.

[0323] To analyze the soluble protein produced, the engineered scaffold protein (S090 (SEQ ID NO: 740)) was passed through a Superdex200 Increase 10 / 300 column by Cytiva using an AKTA FPLC. UV absorbance at 280 nM was recorded as a function of elution volume. For S090, three peaks were recorded at different elution volumes, as seen in Figure 15B. The results in Figure 15B indicate that the engineered scaffold protein was produced as a mixture of monomers, dimers, and multimers, with at least 50% of the protein present as a monomer.

[0324] Example 7. Modified sandwich ELISA assay for protein-peptide interactions Promiscuous epitopes derived from biotin-bearing target proteins were either chemically synthesized or expressed in E. coli and then labeled with biotin using sortase-mediated ligation of short biotinylated peptides.

[0325] The binder protein was coated onto the surface of a high-binding capacity 96-well plate by incubating 100 μL of 5 μg / mL protein in coating solution (10 mM Tris pH 8.8, 50 mM NaCl) overnight at 4°C. The following morning, the coating solution was aspirated, and the wells were washed once with 300 μL of ELISA wash buffer (1× PBS, 0.05% Tween®-20), and the solution was aspirated. The wells were blocked with 200 μL of blocking buffer (1× PBS, 0.05% Tween®-20, 3% BSA) at room temperature for 1 hour. After 1 hour, the blocking solution was aspirated, and 100 μL of biotinylated polypeptide in various concentrations of binding buffer (1× PBS, 0.05% Tween®-20, 0.5% BSA) was added to the appropriate wells and incubated for 20 minutes. After 20 minutes, the peptide solution was aspirated and the wells were washed five times with 300 μL of ELISA wash buffer. To detect the biotinylated peptides, 100 μL of streptavidin-HRP (50 ng / mL) was added to each well and incubated for 1 hour. The wells were then washed five times with 300 μL of ELISA wash buffer. 100 μL of QuantaBlu fluorescent peroxidase substrate solution was added to each well, incubated for 30 minutes, and the reaction was stopped by adding 100 μL of stop solution. The fluorescent signal from each well was measured using a Biotek Cytation 5 plate reader by exciting the fluorophore at 325 nM and measuring the fluorescence at 420 nM. The fluorescent signal was plotted as a function of polypeptide concentration.

[0326] The engineered protein scaffolds S007, S005, and S090 were derived from MHC II molecules, and the binding activity of these engineered scaffolds was measured in an ELISA assay as described herein. The results of increased binding of engineered protein scaffolds S007, S005, and S090 to the disordered regions of CD74, KAAG1, CysLT2, CRACM, and Kv1.5 compared to negative controls are shown in Figures 16A-E. In human cells, all MHC II-based molecules associate with CLIP peptides derived from CD74; therefore, CD74 binding was used as a positive control. The data demonstrate that the engineered scaffold proteins can bind long disordered polypeptides derived from extracellular disease target proteins, including GPCRs (e.g., CysLT2), ion channels (e.g., CRACM, Kv1.5), and membrane proteins (e.g., CD74, KAAG1).

[0327] The results in Figure 17A demonstrate that scaffold optimization and rational design increase binding of the target peptide, which in this assay is CysLT2. In Figure 17A, scaffold v1.0 refers to S007, derived from MHC II, and includes a binding unit (including an 82-amino acid N-terminal alpha chain), a hinge unit (including a 98-amino acid N-terminal beta chain), and an immunoglobulin unit (i.e., a beta-globulin domain), containing the binding groove structure described herein. The binding unit is fused to the N-terminus of the hinge unit, which is fused to the immunoglobulin unit at its C-terminus. Scaffold v1.1 refers to S005, which is derived from S007 by removing the immunoglobulin unit. Scaffold v1.2 refers to S090, which was rationally designed based on v1.1 (S005), and contains amino acid changes that increase the binding efficiency of the target peptide without compromising the binding groove structure.

[0328] Binding affinity, relative binding, and binding specificity relative to a negative control were measured at 10,000 nM of target peptide, and the results can be seen in Figures 17B-D. Fluorescent binding signals were normalized to a control consisting of a binding reaction without any scaffold. Thus, data were obtained from binding isotherms by dividing the fluorescent signal of a particular engineered scaffold protein at that particular target concentration by the fluorescent signal of a control reaction that did not contain any scaffold protein. Figure 17B shows that scaffold optimization and rational design increase the binding affinity of the engineered scaffold protein to the target peptide (CYSLTR2). Similarly, Figures 17C and 17D show that scaffold optimization and rational design increase the relative binding and binding specificity of the engineered scaffold protein to the target peptide (CYSLTR2), respectively.

[0329] Engineered scaffolds S058 and S065 were derived from MHC I proteins. The binding activity of these engineered scaffolds compared to S090, which was derived from MHC II proteins, and a negative control, was measured in an ELISA assay as described herein. Binding results for CD74 (positive control), KAAG1, CRACM, CysLTR2, and Kv1.5 can be seen in Figures 18A-18E.

[0330] Engineered scaffolds S164 and S171 were generated computationally as described herein, and the binding activity of these engineered scaffolds compared to S090 and a negative control was measured in an ELISA assay as described herein. The binding results for CD74 (positive control), KAAG1, CRACM, CysLTR2, and Kv1.5 can be seen in Figures 19A-19E.

[0331] Table 11 summarizes the results of the assays described herein and demonstrates that the engineered scaffold proteins selectively bind to target peptides, even when two or more target peptides can bind, with varying binding strengths. FlhG is a nonspecific hexameric protein used as a positive control to demonstrate that it does not bind to the target of interest. [Table 11] [Table 13]

[0332] Example 8. Selective binding of linear epitopes Next, an ELISA assay, as described in Example 7, was performed to determine the linear epitope of the target irregular region. MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 926) is the N-terminal irregular region of the target CYSLTR2. Naturally occurring MHC molecules typically bind peptides ranging in size from 8 to 20 amino acids. However, certain linear epitopes may exceed 20 amino acids in length. Therefore, narrowing down the target linear epitope is beneficial for enhancing binding activity. In this assay, the approximately 42 amino acid N-terminal irregular region was divided into three N-terminally biotinylated peptides, MERKFMSLQPSISVSEMEPNG (SEQ ID NO: 927), SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928), and TFSNNNSRNCTIENFKREFFP (SEQ ID NO: 929), each approximately 21 AA long and overlapping by approximately 10 AA.

[0333] The binding results for S090 and S177 to three distinct linear epitopes compared to the negative control can be seen in Figures 20A-20D. The results show that targeting SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928) resulted in the greatest increase in binding. S177 was computationally derived from S005 using the methods described herein. The improved binding for S005, S090, and S177 compared to the negative control when targeting the CYSLT2R optimized linear epitope SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928) can be seen in Figure 21.

[0334] Similar assays were performed to compare binding of a short linear epitope (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)) to a longer amino acid sequence from the disordered region of CYSLTR2 (GMERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 930)). Scaffolds assayed included S177, a computationally engineered variant; S189, derived from S177 with an additional dicysteine; wild-type S180; S193, an engineered scaffold protein linked to an N-terminal low-affinity class II-associated invariant chain peptide (CLIP peptide) by a short linker; and S194, an engineered scaffold protein with a low-affinity CLIP peptide with a linker in the middle of the construct. Results compared to negative controls can be seen in Figures 22A-22B.

[0335] Example 9: Binding selectivity of engineered scaffold proteins Next, the binding selectivity of a short linear epitope of CYSLTR2 (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)) was compared to short linear epitopes of KCNA6, TNFRSF13B, KAAG1, CD40LG, and CD74 (positive control). The results can be seen in Figures 23A-23F. Selective binding was observed only for CD74 (positive control) and CYSLTR2.

[0336] Similarly, Table 12 summarizes the results of the assays described herein, presenting heat maps of the engineered scaffold proteins and their binding selectivity to the disordered regions of the target peptides as measured by fluorescent signal intensity. Table 12 demonstrates that the engineered scaffold proteins selectively bind to the target peptides, with varying binding strengths even when more than one target peptide can bind. [Table 12] [Table 14]

[0337] Example 10: BLI assay using OCTET RED Promiscuous epitopes of target proteins bearing biotin were either chemically synthesized or expressed in E. coli and then labeled with biotin using sortase-mediated ligation of short biotinylated peptides.

[0338] Streptavidin sensor chips for the BLI instrument were equilibrated in equilibration buffer (1x PBS, 0.05% Tween® 20, 0.5% BSA) for 5 minutes. 0.5 μM of biotinylated polypeptide in equilibration buffer was allowed to bind to the streptavidin sensor for 60 seconds. After substrate binding, the sensor chip was washed in equilibration buffer for 5 minutes to block nonspecific binding and obtain a baseline reading. The peptide-bound, blocked chip was then introduced into wells containing various concentrations of polypeptide in equilibration buffer. Binding and dissociation data were fitted using OCTET software to obtain k on , k off and the resulting KD, binding affinity.

[0339] The results for S078 and S090, which target the 18-amino acid linear epitope of CLIP, can be seen in Figures 24A and 24B. Figures 25-29 demonstrate that the engineered scaffold proteins, despite being engineered to target the 18-amino acid epitope of CLIP (CD74), are capable of binding to both 18- and 25-amino acid epitopes. This effect is unexpected, as native MHC II molecules are known to bind only short peptides of approximately 18-20 amino acids in length. Furthermore, by comparing the binding of S090 and S005 to a 25-amino acid-long peptide sequence, we observed improved binding of S090 (a repeat form of S005) to the target peptide relative to that of S005 (v1.1), demonstrating that scaffolds have improved binding affinity through optimization and rational design (Figures 25 and 26).

[0340] Example 11: GPCR Binding Engineered scaffold proteins were designed through rational engineering to improve binding affinity for selected target peptides in the GPCR family. The engineered scaffolds included v1.0 (S007), v1.1 (S005), and v1.2 (S090). The engineered scaffold proteins were generated as described in Example 5, and ELISA assays were performed as described in Example 7. Figures 30A and 30B demonstrate that v1.2 exhibits the best performance, demonstrating that the engineered scaffold protein binds to the target peptide with over 65-fold higher affinity compared to a nonspecific protein binder. The best-performing engineered scaffold was assayed to establish selectivity for the GPCR1 target over the related GPCR2 target. The results in Figure 31 demonstrate that v1.2 exhibits binding selectivity for the targeted GPCR over the related GPCR.

[0341] Example 12: Engineered scaffold proteins derived from non-MHC proteins Caspases are proteases that bind to and cleave tetrapeptide sequences following an aspartic acid residue at the C-terminus of a substrate sequence. Their proteolytic activity requires a specific cysteine ​​residue in the catalytic core. Engineered caspase-derived scaffold proteins are generated by mutating the cysteine ​​in the enzyme's active site to alanine or other residues, thereby abolishing the caspase's proteolytic activity. Furthermore, residues in the binding unit of the engineered caspase-derived scaffold protein are altered to increase specificity for specific sequences in the target peptide and to provide high binding affinity. This same approach can be used for any class of proteins that bind to short polypeptide sequences of 4 to 50 amino acids, such as those described herein.

[0342] Example 13: Engineered Scaffold Fusion Proteins An E3 ligase is fused to an engineered scaffold protein at its C-terminus. The engineered scaffold protein is used to specifically bind to disordered regions on target peptides with high affinity and selectivity. When the fusion protein binds to the target protein, the C-terminally fused E3 ligase comes into close contact with the target peptide and can ubiquitinate the target peptide. The ubiquitinated protein is then sequestered and sent to a degradation pathway, for example, by natural cellular mechanisms as described. This same approach can be used to regulate post-translational modifications on target peptides, for example, by fusing kinases, protein phosphatases, etc.

[0343] The steps include producing a soluble form of the fusion protein of interest and separately producing the engineered scaffold protein and the fusion partner (e.g., E3 ligase). The specificity of the engineered scaffold protein-ligase fusion for the target peptide of interest is established using ELISA. Once specificity is established, the activity of the fusion protein is verified using a cell-free ubiquitination assay. To establish activity within cells, the fusion protein is encoded in a mammalian expression plasmid and transfected into mammalian cells (e.g., HEK293), and expression of the fusion protein is confirmed by Western blot analysis. Targeted degradation of the target peptide or protein is determined and verified by transfection and Western blot analysis. The expected result is that the target protein is ubiquitinated in the presence of the fusion protein, but not in the absence of the fusion protein.

[0344] From the foregoing description it will be apparent that changes and modifications can be made to the invention described herein to adapt it to various uses and conditions, and such embodiments also fall within the scope of the following claims.

[0345] The recitation of a series of elements in any definition of a variable herein includes definition of that variable as any single element or combination (or subcombination) of the listed elements. The description of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0346] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. (Item 1) An engineered scaffold protein capable of specifically binding to a disordered region in a target peptide, a. one or more binding domains comprising one or more binding units and one or more hinge units, i. the one or more binding units are capable of binding to the disordered region of the target peptide and comprise one or more amino acid changes compared to a wild-type (WT) counterpart; ii. the one or more hinge units are capable of stabilizing the structure of the binding domain for binding; The binding domain wherein the binding activity of the engineered scaffold protein to the target peptide is increased compared to a WT counterpart of the engineered scaffold. (Item 2) 2. The engineered scaffold protein of item 1, wherein the increased binding activity comprises an increase in binding frequency, binding rate, binding time, binding affinity, or any combination thereof. (Item 3) a. Reduced immunogenicity compared to the corresponding WT; b. Increased solubility compared to the corresponding WT. c. Increased stability compared to the corresponding WT; d. increased or decreased hydrophobicity compared to the corresponding WT; e. increased or decreased hydrophilicity compared to the corresponding WT; f. An increased or decreased surface charge compared to the corresponding WT, or g. Any combination of (a) to (f) 3. The engineered scaffold protein of item 1 or 2, having (Item 4) 4. The engineered scaffold protein of any one of items 1 to 3, wherein the disordered region is located in an internal loop, C-terminal tail, or N-terminal tail of the target peptide. (Item 5) 5. The engineered scaffold protein of any one of items 1 to 4, wherein the disordered region in the target peptide comprises a linear epitope bound by the binding unit. (Item 6) 6. The engineered scaffold protein of item 5, wherein the linear epitope has a length of about 4 to about 40, about 4 to about 30, or about 4 to about 25 amino acids. (Item 7) 7. The engineered scaffold protein of any one of items 1 to 6, wherein the target peptide is comprised in a polypeptide, protein, or protein complex. (Item 8) 8. The engineered scaffold protein of any one of items 1 to 7, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and the length of one protein in the polypeptide, protein, or protein complex is greater than about 30 amino acids. (Item 9) 9. The engineered scaffold protein of any one of items 1 to 8, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and wherein the weight of one protein in the polypeptide, protein, or protein complex is greater than about 100 daltons. (Item 10) 10. The engineered scaffold protein of any one of items 1 to 9, wherein the target peptide is comprised in an extracellular protein. (Item 11) 11. The engineered scaffold protein of any one of items 1 to 10, wherein the target peptide is comprised in a membrane protein. (Item 12) 12. The engineered scaffold protein of any one of items 1 to 11, wherein the target peptide is comprised in an extracellular or membrane protein, including a receptor, an ion channel, or a secreted protein. (Item 13) 13. The engineered scaffold protein of any one of items 1 to 12, wherein the target peptide is comprised in an extracellular protein comprising a GPCR. (Item 14) 14. The engineered scaffold protein of any one of items 1 to 13, wherein the binding unit has an extended structure. (Item 15) 15. The engineered scaffold protein of any one of items 1 to 14, wherein the binding unit, when folded into a tertiary conformation, has a three-dimensional conformation that comprises one or more amino acids that are antisense to one or more amino acids in the disordered region of the target peptide as determined by sense-antisense amino acid pairing. (Item 16) 16. The engineered scaffold protein of any one of items 1 to 15, comprising two binding units. (Item 17) 17. The engineered scaffold protein of any one of items 1 to 16, wherein each of the one or more binding units has about 40 to about 200 amino acids, about 60 to about 150 amino acids, or about 80 to about 100 amino acids. (Item 18) 18. The engineered scaffold protein of any one of items 1 to 17, wherein the one or more binding units each comprise one or more helices, including an alpha helix, a 3.10 helix, and / or a pi helix. (Item 19) 19. The engineered scaffold protein of item 18, wherein the one or more binding units each comprise one or more alpha helices. (Item 20) 20. The engineered scaffold protein of any one of items 1 to 19, wherein the one or more binding units each comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid changes compared to their WT counterparts. (Item 21) 21. The engineered scaffold protein of any one of items 1 to 20, wherein each of the one or more hinge units has about 40 to about 200 amino acids, about 60 to about 150 amino acids, or about 80 to about 100 amino acids. (Item 22) 22. The engineered scaffold protein of any one of items 1 to 21, wherein the one or more hinge units have a concave or partially concave structure. (Item 23) 23. The engineered scaffold protein of any one of items 1 to 22, wherein the one or more hinge units have a partially flexible conformation that can accommodate the disordered region of the target peptide when the disordered region is bound by the engineered scaffold protein. (Item 24) 24. The engineered scaffold protein of any one of items 1 to 23, wherein the one or more hinge units comprise one or more beta sheet strands, a linear peptide, a covalent interaction, a non-covalent interaction, a chemical agent, or any combination thereof. (Item 25) 25. The engineered scaffold protein of item 24, wherein the hinge unit comprises about 3 to about 12 beta sheet protein chains or about 6 to about 10 beta sheet protein chains forming one or more beta sheets. (Item 26) 26. The engineered scaffold protein of item 25, wherein the hinge unit comprises one or two beta sheets. (Item 27) 27. The engineered scaffold protein of any one of items 1 to 26, wherein the one or more hinge units are covalently attached to or linked by one or more linking units to the one or more linking units, or a combination thereof. (Item 28) 28. The engineered scaffold protein of item 27, wherein the one or more linking units are one or more linkers. (Item 29) 29. The engineered scaffold protein of item 28, wherein the one or more linkers comprise a peptide linker. (Item 30) 30. The engineered scaffold protein of any one of items 1 to 29, wherein the one or more linking units and the one or more hinge units are connected as monomers. (Item 31) 31. The engineered scaffold protein of any one of items 1 to 30, wherein the one or more hinge units are attached to the N-terminus or C-terminus of the one or more linking units. (Item 32) 31. The engineered scaffold protein of item 30, wherein the engineered scaffold protein comprises two linkage units and one hinge unit, wherein a first linkage unit is attached to the N-terminus of the hinge unit and a second linkage unit is attached to the C-terminus of the hinge unit. (Item 33) 31. The engineered scaffold protein of item 30, wherein the engineered scaffold protein comprises two linkage units and one hinge unit, wherein a first linkage unit is attached to the N-terminus of a second linkage unit and the hinge unit is attached to the C-terminus of the second linkage unit. (Item 34) 34. The engineered scaffold protein of item 33, wherein the two or more binding units are in an antiparallel structure. (Item 35) 35. The engineered scaffold protein of item 33 or 34, wherein the two or more binding units are hemisymmetric. (Item 36) 27. The engineered scaffold protein of any one of items 1 to 26, wherein the one or more linking units and the one or more hinge units are multimers, and in the presence of the target peptide, the one or more linking units and the one or more hinge units form as a binding domain. (Item 37) 37. The engineered scaffold protein of any one of items 1 to 36, wherein the binding domain is derived from any one of the scaffold proteins listed in Table 1. (Item 38) 38. The engineered scaffold protein of any one of items 1 to 37, wherein the binding domain is a binding groove structure. (Item 39) 39. The engineered scaffold protein of item 38, wherein the binding domain has a clamshell structure. (Item 40) 40. The engineered scaffold protein of any one of items 1 to 39, further comprising one or more immunoglobulin units. (Item 41) 41. The engineered scaffold protein of item 40, comprising two immunoglobulin units. (Item 42) 41. The engineered scaffold protein of item 39 or 40, wherein the one or more immunoglobulin units are covalently attached or attached by a linking unit to the binding domain, the hinge unit, the binding unit, or a combination thereof. (Item 43) 40. The engineered scaffold protein of any one of items 1 to 39, which does not comprise an immunoglobulin unit. (Item 44) 44. The engineered scaffold protein of any one of items 1 to 43, having an isoelectric point of 3.5 to 9, 4 to 8.5, or 4.5 to 8 as measured in an electrophoresis assay. (Item 45) 45. The engineered scaffold protein of any one of items 1 to 44, wherein the binding unit comprises an amino acid sequence that is at least 65% identical to any one of the sequences in Table 4. (Item 46) 46. ​​The engineered scaffold protein of any one of items 1 to 45, wherein the hinge unit comprises an amino acid sequence that is at least 65% identical to any one of the sequences in Table 5. (Item 47) 47. The engineered scaffold protein of any one of items 1 to 46, wherein the disordered region in the target peptide comprises an amino acid sequence selected from the list of sequences of bound peptides listed in Table 3, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes or more. (Item 48) 48. The engineered scaffold protein of any one of items 1 to 47, wherein the engineered protein scaffold is conjugated to a heterologous agent to extend the half-life of the engineered scaffold. (Item 49) 49. The engineered scaffold protein of item 48, wherein the heterologous agent is selected from the group consisting of polyethylene glycol (PEG), human serum albumin (HSA), and a variant Fc region of an antibody. (Item 50) 50. The engineered scaffold protein of item 48 or 49, wherein the half-life of the engineered protein scaffold is extended compared to a WT counterpart. (Item 51) 51. The engineered scaffold protein of any one of items 1 to 50, wherein said disordered region in the target peptide comprises an amino acid sequence selected from the list of sequences of bound peptides listed in Table 7, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes or more. (Item 52) 52. The engineered scaffold protein of any one of items 1 to 51, further comprising one or more epitope masking units. (Item 53) 53. The engineered scaffold protein of item 52, wherein the epitope masking unit interacts with the binding unit of the engineered scaffold protein. (Item 54) 54. The engineered scaffold protein of item 52 or 53, wherein the epitope masking unit is linked to the N-terminus, C-terminus, or a mid-loop of the engineered scaffold protein. (Item 55) 55. The engineered scaffold protein of any one of items 52 to 54, wherein the epitope masking unit is linked to the N-terminus, the C-terminus, or a mid-loop of the engineered scaffold protein. (Item 56) 56. The engineered scaffold protein of any one of items 52 to 55, wherein the epitope masking unit is capable of partially masking the binding unit, supporting the tertiary conformation of the engineered scaffold protein, improving the immunogenicity of the engineered scaffold protein, or any combination thereof. (Item 57) 57. The engineered scaffold protein of any one of items 52 to 56, wherein upon binding of the binding unit, an epitope masking unit replaces the disordered region of the target peptide. (Item 58) 58. The engineered scaffold protein of any one of items 1 to 57, having a functional class selected from the list of functional classes of human protein scaffolds listed in Table 2. (Item 59) 59. The engineered scaffold protein of any one of items 1 to 58, comprising an amino acid sequence that is at least 65% identical to any one of the sequences listed in Table 6. (Item 60) 60. The engineered scaffold protein of any one of items 1 to 59, comprising an amino acid sequence that is at least 75% identical to any one of the sequences listed in Table 6. (Item 61) 61. The engineered scaffold protein of any one of items 1 to 60, comprising an amino acid sequence that is at least 80% identical to any one of the sequences listed in Table 6. (Item 62) 62. The engineered scaffold protein of any one of items 1 to 61, comprising an amino acid sequence that is at least 85% identical to any one of the sequences listed in Table 6. (Item 63) 63. The engineered scaffold protein of any one of items 1 to 62, comprising an amino acid sequence that is at least 95% identical to any one of the sequences listed in Table 6. (Item 64) 64. The engineered scaffold protein of any one of items 1 to 63, comprising an amino acid sequence that is at least 97% identical to any one of the sequences listed in Table 6. (Item 65) 65. The engineered scaffold protein of any one of items 1 to 64, comprising an amino acid sequence that is at least 98% identical to any one of the sequences listed in Table 6. (Item 66) 66. The engineered scaffold protein of any one of items 1 to 65, comprising an amino acid sequence that is at least 99% identical to any one of the sequences listed in Table 6. (Item 67) 67. The engineered scaffold protein of any one of items 1 to 66, comprising an amino acid sequence identical to any one of the sequences set forth in Table 6. (Item 68) 68. A fusion protein comprising the engineered scaffold of any one of items 1 to 67 and a fusion partner. (Item 69) 69. The fusion protein of item 68, wherein the fusion partner is an enzyme. (Item 70) 70. The fusion protein of item 69, wherein the enzyme catalyzes ubiquitination, post-translational modification, proteolytic cleavage, dephosphorylation, trans-cis isomerization, protein chaperone activity, nucleic acid-modifying protein, ATPase or GTPase activity. (Item 71) 71. The fusion protein of any one of items 68 to 70, wherein the fusion partner selectively binds to a specific region on the target protein. (Item 72) 72. The fusion protein of item 71, wherein the specific region on the target protein is not a disordered region of the target protein. (Item 73) 68. A method for binding to a disordered region of a target protein, the method comprising contacting a target protein comprising said disordered region with the engineered scaffold protein of any one of items 1 to 67. (Item 74) 73. A method for binding to an irregular region of a target protein, comprising contacting a target protein comprising said irregular region with the fusion protein of any one of items 68 to 72. (Item 75) 68. A method for altering the conformation of a protein comprising a target peptide, the method comprising contacting the target peptide with the engineered scaffold protein of any one of items 1 to 67. (Item 76) 73. A method for altering the conformation of a protein comprising a target peptide, the method comprising contacting the target peptide with the fusion protein of any one of items 68 to 72. (Item 77) 68. A method of treating a disease, comprising administering to a subject an effective amount of the engineered scaffold protein of any one of items 1 to 67. (Item 78) 73. A method for treating a disease, comprising administering to a subject an effective amount of the fusion protein of any one of items 68 to 72. (Item 79) 68. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject the engineered scaffold protein of any one of items 1 to 67. (Item 80) 73. A method for inducing an immune response in a subject in need thereof, comprising administering to the subject the fusion protein of any one of items 68 to 72. (Item 81) 68. A method for detecting a target protein, the method comprising contacting a sample suspected of containing the target protein with at least the engineered scaffold protein of any one of items 1 to 67. (Item 82) 73. A method for detecting a target protein, comprising contacting a sample suspected of containing the target protein with at least the fusion protein of any one of items 68 to 72. (Item 83) 68. A kit comprising at least the engineered scaffold protein of any one of items 1 to 67. (Item 84) 73. A kit comprising at least the fusion protein of any one of items 68 to 72. (Item 85) 68. A device comprising at least the engineered scaffold protein of any one of items 1 to 67. (Item 86) 73. A device comprising at least the fusion protein of any one of items 68 to 72. (Item 87) 68. A method for detecting a compound in a sample, the method comprising contacting the sample with an engineered scaffold protein according to any one of items 1 to 67. (Item 88) 73. A method for detecting a compound in a sample, comprising contacting the sample with a fusion protein according to any one of items 68 to 72. (Item 89) 68. A pharmaceutical composition comprising the engineered scaffold protein of any one of items 1 to 67 and a pharmaceutically acceptable excipient. (Item 90) 73. A pharmaceutical composition comprising the fusion protein of any one of items 68 to 72 and a pharmaceutically acceptable excipient. (Item 91) A method for preventing, treating, or maintaining a disease in a subject in need thereof by administering an effective amount of the pharmaceutical composition according to item 32. (Item 92) A method for preventing, treating, or maintaining a disease in a subject in need thereof by administering an effective amount of the pharmaceutical composition according to item 33. (Item 93) 68. An isolated nucleic acid molecule encoding the engineered scaffold protein of any one of items 1 to 67. (Item 94) 73. An isolated nucleic acid molecule encoding the fusion protein of any one of items 68 to 72. (Item 95) 94. An expression vector operably linked to the nucleic acid of Item 93. (Item 96) 95. An expression vector operably linked to the nucleic acid of Item 94. (Item 97) A host cell comprising the vector described in Item 95. (Item 98) A host cell comprising the vector described in Item 96. (Item 99) 68. A polypeptide display library comprising the engineered scaffold protein of any one of items 1 to 67. (Item 100) 73. A polypeptide display library comprising the fusion protein of any one of items 68 to 72. (Item 101) 100. The polypeptide display library of claim 99, wherein the engineered scaffold protein is displayed on the surface of a virus or yeast, or as a ribosomal or RNA-conjugated protein molecule. (Item 102) 101. The polypeptide display library of claim 100, wherein the fusion protein is displayed on the surface of a virus or yeast, or as a ribosome or RNA-conjugated protein molecule. (Item 103) 99. A collection of isolated nucleic acid molecules encoding the library of item 99. (Item 104) A collection of isolated nucleic acid molecules encoding the library of item 100. (Item 105) 100. A method for obtaining an engineered scaffold that binds to a target, the method comprising: (a) contacting a target ligand with the library of claim 99 under conditions that allow the formation of an engineered scaffold protein:target ligand complex; and (b) obtaining from the complex the scaffold that binds to the target ligand. (Item 106) 102. A method for obtaining a fusion protein that binds to a target, the method comprising: (a) contacting a target ligand with the library of claim 100 under conditions that allow the formation of a fusion protein:target ligand complex; and (b) obtaining from the complex the fusion protein that binds to the target ligand. (Item 107) 100. A method for obtaining at least two engineered scaffold proteins that bind to a target, the method comprising: (a) contacting a target ligand with the library of claim 99 under conditions that allow the formation of an engineered scaffold:target ligand complex; (b) binding the complex with a cross-linking agent, wherein cross-linking of the complex elicits a detectable response; and (c) obtaining from the complex the engineered scaffold proteins that bind to the target. (Item 108) 108. The method of claim 107, wherein the engineered scaffold proteins recognize the same epitope. (Item 109) 108. The method of claim 107, wherein the engineered scaffold proteins recognize distinct epitopes. (Item 110) 108. The method of claim 107, wherein the cross-linking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag. (Item 111) 100. A method for obtaining at least two fusion proteins that bind to a target, the method comprising: (a) contacting a target ligand with the library of claim 100 under conditions that allow the formation of a fusion protein:target ligand complex; (b) binding the complex with a cross-linking agent, wherein cross-linking of the complex elicits a detectable response; and (c) obtaining from the complex the fusion proteins that bind to the target. (Item 112) 112. The method of claim 111, wherein the fusion proteins recognize the same epitope. (Item 113) 112. The method of claim 111, wherein the fusion proteins recognize distinct epitopes. (Item 114) 112. The method of claim 111, wherein the cross-linking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag. (Item 115) 1. A method for generating an engineered scaffold protein sequence capable of binding to a disordered region of a target peptide, comprising: a. selecting one or more scaffold protein sequences; b. evaluating the one or more scaffold protein sequences for one or more desired characteristics including ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, or any combination thereof; c. engineering the amino acid sequence of an engineered scaffold protein based on said evaluation of said one or more scaffold protein sequences. wherein the engineered scaffold protein is predicted to have enhanced or improved one or more desired characteristics compared to one or more scaffold protein counterparts. (Item 116) 116. The method of claim 115, wherein engineering the amino acid sequence of the engineered scaffold protein comprises engineering the amino acid sequence to bind to a linear epitope of the target peptide. (Item 117) 117. The method of claim 115 or 116, wherein evaluating the one or more scaffold protein sequences for one or more desired characteristics comprises weighting one or more factors of the one or more scaffold protein sequences that are related to one or more desired characteristics, wherein the one or more weighted factors comprise three-dimensional structure, protein domain(s), amino acid sequence, amino acid charge, amino acid polarity, amino acid hydrophobicity / hydrophilicity, amino acid acidity / basicity, or any combination thereof. (Item 118) Weighting one or more factors may a. assigning values ​​to the one or more weighted factors based on an estimated probability of improving one or more desired characteristics; b. assigning a value to said one or more weighted factors and measuring the deviation of said value from a target value or from a value assigned to said factor of a second scaffold protein; or c. Both (a) and (b) Item 118. The method of item 117, comprising: (Item 119) a. whether the engineered amino acid sequence represents an engineered scaffold protein that exhibits improved characteristics; b. whether a change in one or more of said one or more weighted factors improves said value compared to a target value or threshold or a value assigned to such factor of a second scaffold protein; or c. Both (a) and (b) Item 119. The method of item 118, further comprising predicting: (Item 120) 120. The method of claim 119, wherein the one or more changes comprise one or more amino acid changes. (Item 121) 121. The method of claim 119(b) or 120, wherein the engineered amino acid sequence of the engineered scaffold protein comprises one or more changes compared to a counterpart scaffold protein. (Item 122) 122. The method of any one of items 115 to 121, wherein evaluating the one or more scaffold protein sequences comprises manually evaluating the amino acid sequences of the one or more scaffold protein sequences or evaluating the amino acid sequences of the one or more scaffold protein sequences in a machine learning model. (Item 123) a. assessing another desired characteristic of the engineered amino acid sequence of the engineered scaffold protein; b. weighting one or more weighting factors of the engineered amino acid sequence of the engineered scaffold protein; or c. Both (a) and (b) and informing further manipulation of the amino acid sequence of the engineered scaffold protein. (Item 124) 124. The method of any one of items 115 to 123, comprising producing the engineered scaffold protein. (Item 125) 125. The method of any one of items 115 to 124, comprising assaying the engineered scaffold protein. (Item 126) 126. The method of claim 125, wherein the assaying comprises one or more in vitro or in vivo assays. (Item 127) 127. The method of claim 125 or 126, wherein assaying comprises in silico simulation and / or machine learning model simulation. (Item 128) 128. The method of claim 126 or 127, wherein assaying the produced engineered scaffold protein comprises obtaining data and providing information for further production of engineered scaffold protein. (Item 129) A system comprising instructions capable of carrying out the method according to any one of items 115 to 123 and 125 to 127. (Item 130) 129. An engineered scaffold protein comprising an amino acid sequence produced by the method of any one of items 115 to 128 or the system of item 129. (Item 131) 73. A composition comprising the engineered scaffold protein of any one of items 1 to 67 or the fusion protein of any one of items 68 to 72 and a therapeutic partner. (Item 132) 132. The composition of claim 131, wherein the therapeutic partner is attached to the engineered scaffold protein or the fusion protein, and the therapeutic partner is covalently bound, non-covalently bound, fused, conjugated, and / or linked to the engineered scaffold protein or the fusion protein. (Item 133) 133. The composition of claim 131 or 132, wherein the therapeutic partner comprises one or more of an anti-cancer agent, an anti-inflammatory agent, an antibacterial agent, an antiviral agent, a cytokine, a toxin, an enzyme, a neuroprotective agent, a soluble factor scavenger, or any combination thereof. (Item 134) 134. The composition of claim 132 or 133, wherein the therapeutic partner can be separated from the engineered scaffold protein or the fusion protein upon binding to the target peptide. (Item 135) 135. The composition of any one of items 131 to 134, wherein the engineered scaffold protein or the fusion protein can be tissue-specific or cell-specific. (Item 136) 135. The composition of claim 134, wherein the therapeutic partner can be separated from the engineered scaffold protein or the fusion protein upon reaching a specific cell or tissue. (Item 137) 137. A method for treating a disease or disorder, comprising administering to a subject in need thereof the composition of any one of items 131 to 136. (Item 138) 1. A method of chimeric antigen receptor (CAR) T cell therapy comprising administering to a subject in need thereof: a. the engineered scaffold protein of any one of items 1 to 67, or b. The fusion protein according to any one of items 68 to 72. The method comprises administering

Claims

1. An engineered scaffold protein capable of specifically binding to a disordered region in a target peptide, a. one or more binding domains comprising one or more binding units and one or more hinge units, i. the one or more binding units are capable of binding to the disordered region of the target peptide and comprise one or more amino acid changes compared to a wild-type (WT) counterpart; ii. the one or more hinge units are capable of stabilizing the structure of the binding domain for binding; The binding domain wherein the binding activity of the engineered scaffold protein to the target peptide is increased compared to a WT counterpart of the engineered scaffold.

2. 2. The engineered scaffold protein of claim 1, wherein the increased binding activity comprises an increase in binding frequency, binding rate, binding time, binding affinity, or any combination thereof.

3. a. reduced immunogenicity compared to the corresponding WT; b. Increased solubility compared to the corresponding WT; c. Increased stability compared to the corresponding WT; d. increased or decreased hydrophobicity compared to the corresponding WT; e. increased or decreased hydrophilicity compared to the corresponding WT; f. An increased or decreased surface charge compared to the corresponding WT, or g. Any combination of (a) to (f) 3. The engineered scaffold protein of claim 1 or 2, having

4. 4. The engineered scaffold protein of claim 1, wherein the disordered region is located in an internal loop, a C-terminal tail, or an N-terminal tail of the target peptide.

5. 5. The engineered scaffold protein of claim 1, wherein the disordered region in the target peptide comprises a linear epitope bound by the binding unit.

6. 6. The engineered scaffold protein of claim 5, wherein said linear epitope has a length of about 4 to about 40, about 4 to about 30, or about 4 to about 25 amino acids.

7. 7. The engineered scaffold protein of claim 1 , wherein the target peptide is comprised in a polypeptide, protein, or protein complex.

8. 8. The engineered scaffold protein of any one of claims 1 to 7, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and wherein the length of one protein in the polypeptide, protein, or protein complex is greater than about 30 amino acids.

9. 9. The engineered scaffold protein of any one of claims 1 to 8, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and wherein the weight of one protein in the polypeptide, protein, or protein complex is greater than about 100 daltons.

10. 10. The engineered scaffold protein of claim 1, wherein the target peptide is comprised in an extracellular protein.

11. The engineered scaffold protein of claim 1 , wherein the target peptide is comprised in a membrane protein.

12. 12. The engineered scaffold protein of any one of claims 1 to 11, wherein the target peptide is comprised in an extracellular or membrane protein, including a receptor, an ion channel, or a secreted protein.

13. 13. The engineered scaffold protein of any one of claims 1 to 12, wherein the target peptide is comprised in an extracellular protein comprising a GPCR.

14. 14. The engineered scaffold protein of claim 1, wherein the binding unit has an extended structure.

15. 15. The engineered scaffold protein of any one of claims 1 to 14, wherein the binding unit, when folded into its tertiary conformation, has a three-dimensional conformation that comprises one or more amino acids that are antisense to one or more amino acids in the disordered region of the target peptide as determined by sense-antisense amino acid pairing.

16. 16. The engineered scaffold protein of any one of claims 1 to 15, comprising two binding units.

17. 17. The engineered scaffold protein of any one of claims 1 to 16, wherein each of the one or more binding units has from about 40 to about 200 amino acids, from about 60 to about 150 amino acids, or from about 80 to about 100 amino acids.

18. 18. The engineered scaffold protein of any one of claims 1 to 17, wherein the one or more binding units each comprise one or more helices, including an alpha helix, a 3.10 helix, and / or a pi helix.

19. 20. The engineered scaffold protein of claim 18, wherein said one or more binding units each comprise one or more alpha helices.

20. 20. The engineered scaffold protein of any one of claims 1 to 19, wherein said one or more binding units each comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid changes compared to their WT counterparts.

21. 21. The engineered scaffold protein of any one of claims 1 to 20, wherein each of the one or more hinge units has from about 40 to about 200 amino acids, from about 60 to about 150 amino acids, or from about 80 to about 100 amino acids.

22. 22. The engineered scaffold protein of any one of claims 1 to 21, wherein the one or more hinge units have a concave or partially concave structure.

23. 23. The engineered scaffold protein of any one of claims 1 to 22, wherein the one or more hinge units have a partially flexible conformation that can accommodate the disordered region of the target peptide when the disordered region is bound by the engineered scaffold protein.

24. 24. The engineered scaffold protein of any one of claims 1 to 23, wherein the one or more hinge units comprise one or more beta sheet strands, linear peptides, covalent interactions, non-covalent interactions, chemical agents, or any combination thereof.

25. 25. The engineered scaffold protein of Claim 24, wherein the hinge unit comprises about 3 to about 12 beta sheet protein chains or about 6 to about 10 beta sheet protein chains forming one or more beta sheets.

26. 26. The engineered scaffold protein of claim 25, wherein the hinge unit comprises one or two beta sheets.

27. 27. The engineered scaffold protein of any one of claims 1 to 26, wherein the one or more hinge units are covalently attached to or linked by one or more linking units to the one or more linking units, or a combination thereof.

28. 28. The engineered scaffold protein of claim 27, wherein said one or more linking units are one or more linkers.

29. 30. The engineered scaffold protein of claim 28, wherein said one or more linkers comprise a peptide linker.

30. 30. The engineered scaffold protein of any one of claims 1 to 29, wherein the one or more linking units and the one or more hinge units are connected as monomers.

31. 31. The engineered scaffold protein of any one of claims 1 to 30, wherein the one or more hinge units are attached to the N-terminus or C-terminus of the one or more linking units.

32. 31. The engineered scaffold protein of claim 30, wherein the engineered scaffold protein comprises two linkage units and one hinge unit, wherein a first linkage unit is attached to the N-terminus of the hinge unit and a second linkage unit is attached to the C-terminus of the hinge unit.

33. 31. The engineered scaffold protein of claim 30, wherein the engineered scaffold protein comprises two linkage units and one hinge unit, wherein a first linkage unit is attached to the N-terminus of a second linkage unit and said hinge unit is attached to the C-terminus of said second linkage unit.

34. 34. The engineered scaffold protein of claim 33, wherein the two or more binding units are in an antiparallel structure.

35. 35. The engineered scaffold protein of claim 33 or 34, wherein the two or more binding units are hemisymmetric.

36. 27. The engineered scaffold protein of any one of claims 1 to 26, wherein the one or more linking units and the one or more hinge units are multimers, and in the presence of the target peptide, the one or more linking units and the one or more hinge units form as a binding domain.

37. 37. The engineered scaffold protein of any one of claims 1 to 36, wherein the binding domain is derived from any one of the scaffold proteins listed in Table 1.

38. 38. The engineered scaffold protein of any one of claims 1 to 37, wherein the binding domain is a binding groove structure.

39. 39. The engineered scaffold protein of claim 38, wherein said binding domain has a clamshell structure.

40. 40. The engineered scaffold protein of any one of claims 1 to 39, further comprising one or more immunoglobulin units.

41. 41. The engineered scaffold protein of claim 40, comprising two immunoglobulin units.

42. 42. The engineered scaffold protein of claim 40 or 41, wherein the one or more immunoglobulin units are covalently attached or attached by a linking unit to the binding domain, the hinge unit, the binding unit, or a combination thereof.

43. 40. The engineered scaffold protein of any one of claims 1 to 39, which does not comprise an immunoglobulin unit.

44. 44. The engineered scaffold protein of any one of claims 1 to 43, having an isoelectric point of 3.5 to 9, 4 to 8.5, or 4.5 to 8 as measured in an electrophoresis assay.

45. 45. The engineered scaffold protein of any one of claims 1 to 44, wherein the binding unit comprises an amino acid sequence that is at least 65% identical to any one of the sequences in Table 4.

46. 46. ​​The engineered scaffold protein of any one of claims 1 to 45, wherein the hinge unit comprises an amino acid sequence that is at least 65% identical to any one of the sequences in Table 5.

47. 47. The engineered scaffold protein of any one of claims 1 to 46, wherein said disordered region in the target peptide comprises an amino acid sequence selected from the list of sequences of bound peptides listed in Table 3, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid changes.

48. 48. The engineered scaffold protein of any one of claims 1 to 47, wherein the engineered protein scaffold is conjugated to a heterologous agent to extend the half-life of the engineered scaffold.

49. 49. The engineered scaffold protein of claim 48, wherein the heterologous agent is selected from the group consisting of polyethylene glycol (PEG), human serum albumin (HSA), and a variant Fc region of an antibody.

50. 50. The engineered scaffold protein of claim 48 or 49, wherein the half-life of the engineered protein scaffold is extended compared to its WT counterpart.

51. 51. The engineered scaffold protein of any one of claims 1 to 50, wherein said disordered region in the target peptide comprises an amino acid sequence selected from the list of sequences of bound peptides listed in Table 7, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid changes.

52. 52. The engineered scaffold protein of any one of claims 1 to 51, further comprising one or more epitope masking units.

53. 53. The engineered scaffold protein of claim 52, wherein said epitope masking unit interacts with said binding unit of said engineered scaffold protein.

54. 54. The engineered scaffold protein of claim 52 or 53, wherein the epitope masking unit is linked to the N-terminus, C-terminus, or a mid-loop of the engineered scaffold protein.

55. 55. The engineered scaffold protein of any one of claims 52 to 54, wherein the epitope masking unit is linked to the N-terminus, the C-terminus, or a mid-loop of the engineered scaffold protein.

56. 56. The engineered scaffold protein of any one of claims 52 to 55, wherein the epitope masking unit is capable of partially masking the binding unit, supporting the tertiary structure of the engineered scaffold protein, improving the immunogenicity of the engineered scaffold protein, or any combination thereof.

57. 57. The engineered scaffold protein of any one of claims 52 to 56, wherein upon binding of the binding unit, an epitope masking unit replaces the disordered region of the target peptide.

58. 58. The engineered scaffold protein of any one of claims 1 to 57, having a functional class selected from the list of functional classes of human protein scaffolds listed in Table 2.

59. 59. The engineered scaffold protein of any one of claims 1 to 58, comprising an amino acid sequence that is at least 65% identical to any one of the sequences set forth in Table 6.

60. 60. The engineered scaffold protein of any one of claims 1 to 59, comprising an amino acid sequence that is at least 75% identical to any one of the sequences listed in Table 6.

61. 61. The engineered scaffold protein of any one of claims 1 to 60, comprising an amino acid sequence that is at least 80% identical to any one of the sequences set forth in Table 6.

62. 62. The engineered scaffold protein of any one of claims 1 to 61, comprising an amino acid sequence that is at least 85% identical to any one of the sequences set forth in Table 6.

63. 63. The engineered scaffold protein of any one of claims 1 to 62, comprising an amino acid sequence that is at least 95% identical to any one of the sequences listed in Table 6.

64. 64. The engineered scaffold protein of any one of claims 1 to 63, comprising an amino acid sequence that is at least 97% identical to any one of the sequences set forth in Table 6.

65. 65. The engineered scaffold protein of any one of claims 1 to 64, comprising an amino acid sequence that is at least 98% identical to any one of the sequences set forth in Table 6.

66. 66. The engineered scaffold protein of any one of claims 1 to 65, comprising an amino acid sequence that is at least 99% identical to any one of the sequences set forth in Table 6.

67. 67. The engineered scaffold protein of any one of claims 1 to 66, comprising an amino acid sequence identical to any one of the sequences set forth in Table 6.

68. 68. A fusion protein comprising the engineered scaffold of any one of claims 1 to 67 and a fusion partner.

69. 69. The fusion protein of claim 68, wherein the fusion partner is an enzyme.

70. 70. The fusion protein of claim 69, wherein the enzyme catalyzes ubiquitination, post-translational modification, proteolytic cleavage, dephosphorylation, trans-cis isomerization, protein chaperone activity, nucleic acid-modifying protein, ATPase, or GTPase activity.

71. 71. A fusion protein according to any one of claims 68 to 70, wherein the fusion partner selectively binds to a specific region on the target protein.

72. 72. The fusion protein of claim 71, wherein the specific region on the target protein is not a disordered region of the target protein.

73. 68. A method for binding to a disordered region of a target protein, the method comprising contacting a target protein comprising said disordered region with an engineered scaffold protein of any one of claims 1 to 67.

74. 73. A method for binding to an irregular region of a target protein, the method comprising contacting a target protein comprising said irregular region with a fusion protein of any one of claims 68 to 72.

75. 68. A method for altering the conformation of a protein comprising a target peptide, the method comprising contacting the target peptide with an engineered scaffold protein described in any one of claims 1 to 67.

76. 73. A method for altering the conformation of a protein comprising a target peptide, the method comprising contacting the target peptide with a fusion protein described in any one of claims 68 to 72.

77. 100. A method of treating a disease, comprising administering to a subject an effective amount of the engineered scaffold protein of any one of claims 1-67.

78. 73. A method of treating a disease, comprising administering to a subject an effective amount of a fusion protein of any one of claims 68 to 72.

79. 100. A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject the engineered scaffold protein of any one of claims 1 to 67.

80. 73. A method for inducing an immune response in a subject in need thereof, comprising administering to said subject a fusion protein of any one of claims 68 to 72.

81. 68. A method for detecting a target protein, the method comprising contacting a sample suspected of containing the target protein with at least the engineered scaffold protein of any one of claims 1 to 67.

82. 73. A method for detecting a target protein, the method comprising contacting a sample suspected of containing the target protein with at least the fusion protein of any one of claims 68 to 72.

83. 68. A kit comprising at least the engineered scaffold protein of any one of claims 1 to 67.

84. 73. A kit comprising at least a fusion protein according to any one of claims 68 to 72.

85. 68. A device comprising at least the engineered scaffold protein of any one of claims 1 to 67.

86. 73. A device comprising at least a fusion protein according to any one of claims 68 to 72.

87. 68. A method for detecting a compound in a sample, the method comprising contacting the sample with an engineered scaffold protein of any one of claims 1 to 67.

88. 73. A method for detecting a compound in a sample, the method comprising contacting the sample with a fusion protein of any one of claims 68 to 72.

89. 68. A pharmaceutical composition comprising the engineered scaffold protein of any one of claims 1 to 67 and a pharmaceutically acceptable excipient.

90. 73. A pharmaceutical composition comprising the fusion protein of any one of claims 68 to 72 and a pharmaceutically acceptable excipient.

91. 90. A method of preventing, treating, or maintaining a disease in a subject in need thereof by administering an effective amount of the pharmaceutical composition of claim 89.

92. 91. A method of preventing, treating, or maintaining a disease in a subject in need thereof by administering an effective amount of the pharmaceutical composition of claim 90.

93. 68. An isolated nucleic acid molecule encoding the engineered scaffold protein of any one of claims 1 to 67.

94. 73. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 68 to 72.

95. 94. An expression vector operably linked to the nucleic acid of claim 93.

96. 95. An expression vector operably linked to the nucleic acid of claim 94.

97. A host cell comprising the vector of claim 95.

98. 97. A host cell comprising the vector of claim 96.

99. 68. A polypeptide display library comprising the engineered scaffold protein of any one of claims 1 to 67.

100. 73. A polypeptide display library comprising the fusion protein of any one of claims 68 to 72.

101. 100. The polypeptide display library of claim 99, wherein the engineered scaffold protein is displayed on the surface of a virus or yeast, or is displayed as a ribosome or RNA-conjugated protein molecule.

102. 101. The polypeptide display library of claim 100, wherein the fusion protein is displayed on the surface of a virus or yeast, or displayed as a ribosome or RNA-conjugated protein molecule.

103. 100. A collection of isolated nucleic acid molecules encoding the library of claim 99.

104. 101. A collection of isolated nucleic acid molecules encoding the library of claim 100.

105. 100. A method for obtaining an engineered scaffold that binds to a target, the method comprising: (a) contacting a target ligand with the library of claim 99 under conditions that allow the formation of an engineered scaffold protein:target ligand complex; and (b) obtaining from the complex the scaffold that binds to the target ligand.

106. 101. A method for obtaining a fusion protein that binds to a target, the method comprising: (a) contacting a target ligand with the library of claim 100 under conditions that allow the formation of a fusion protein:target ligand complex; and (b) obtaining from the complex the fusion protein that binds to the target ligand.

107. 100. A method for obtaining at least two engineered scaffold proteins that bind to a target, the method comprising: (a) contacting a target ligand with the library of claim 99 under conditions that allow the formation of an engineered scaffold:target ligand complex; (b) binding the complex with a cross-linking agent, wherein cross-linking of the complex elicits a detectable response; and (c) obtaining from the complex the engineered scaffold proteins that bind to the target.

108. 108. The method of claim 107, wherein the engineered scaffold proteins recognize the same epitope.

109. 108. The method of claim 107, wherein the engineered scaffold proteins recognize distinct epitopes.

110. 108. The method of claim 107, wherein the cross-linking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag.

111. 101. A method for obtaining at least two fusion proteins that bind to a target, the method comprising: (a) contacting a target ligand with the library of claim 100 under conditions that allow the formation of a fusion protein:target ligand complex; (b) binding the complex with a cross-linking agent, wherein cross-linking of the complex elicits a detectable response; and (c) obtaining from the complex the fusion proteins that bind to the target.

112. The method of claim 111, wherein the fusion proteins recognize the same epitope.

113. 112. The method of claim 111, wherein the fusion proteins recognize distinct epitopes.

114. 112. The method of claim 111, wherein the cross-linking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag.

115. 1. A method for generating an engineered scaffold protein sequence capable of binding to a disordered region of a target peptide, comprising: a. selecting one or more scaffold protein sequences; b. evaluating the one or more scaffold protein sequences for one or more desired characteristics including ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding time, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, or any combination thereof; c. engineering the amino acid sequence of an engineered scaffold protein based on said evaluation of said one or more scaffold protein sequences. wherein the engineered scaffold protein is predicted to have enhanced or improved one or more desired characteristics compared to one or more scaffold protein counterparts.

116. 116. The method of claim 115, wherein engineering the amino acid sequence of the engineered scaffold protein comprises engineering the amino acid sequence to bind to a linear epitope of the target peptide.

117. 117. The method of claim 115 or 116, wherein evaluating the one or more scaffold protein sequences for one or more desired characteristics comprises weighting one or more factors of the one or more scaffold protein sequences that are associated with one or more desired characteristics, wherein the one or more weighted factors comprise three-dimensional structure, protein domain(s), amino acid sequence, amino acid charge, amino acid polarity, amino acid hydrophobicity / hydrophilicity, amino acid acidity / basicity, or any combination thereof.

118. Weighting one or more factors a. assigning values ​​to the one or more weighted factors based on an estimated probability of improving one or more desired characteristics; b. assigning a value to said one or more weighted factors and measuring the deviation of said value from a target value or from a value assigned to said factor of a second scaffold protein; or c. Both (a) and (b) 118. The method of claim 117, comprising:

119. a. whether the engineered amino acid sequence represents an engineered scaffold protein that exhibits improved characteristics; b. whether a change in one or more of said weighted factors improves said value compared to a target value or threshold or a value assigned to such factor of a second scaffold protein; or c. Both (a) and (b) 119. The method of claim 118, further comprising predicting:

120. 120. The method of claim 119, wherein the one or more changes comprise one or more amino acid changes.

121. 121. The method of claim 119(b) or 120, wherein the engineered amino acid sequence of the engineered scaffold protein comprises one or more changes compared to a counterpart scaffold protein.

122. 122. The method of any one of claims 115-121, wherein evaluating the one or more scaffold protein sequences comprises manually evaluating the amino acid sequences of the one or more scaffold protein sequences or evaluating the amino acid sequences of the one or more scaffold protein sequences in a machine learning model.

123. a. assessing another desired characteristic of the engineered amino acid sequence of the engineered scaffold protein; b. weighting one or more weighting factors of the engineered amino acid sequences of the engineered scaffold protein; or c. Both (a) and (b) and informing further manipulation of the amino acid sequence of the engineered scaffold protein.

124. 124. The method of any one of claims 115 to 123, comprising producing said engineered scaffold protein.

125. 125. The method of any one of claims 115 to 124, comprising assaying the engineered scaffold protein.

126. 126. The method of claim 125, wherein the assaying comprises one or more in vitro or in vivo assays.

127. 127. The method of claim 125 or 126, wherein the assaying comprises in silico simulation and / or machine learning model simulation.

128. 128. The method of claims 126 or 127, wherein assaying the produced engineered scaffold protein comprises obtaining data and providing information for further production of engineered scaffold protein.

129. 128. A system comprising instructions capable of carrying out the method of any one of claims 115 to 123 and 125 to 127.

130. 130. An engineered scaffold protein comprising an amino acid sequence produced by the method of any one of claims 115 to 128 or the system of claim 129.

131. 73. A composition comprising the engineered scaffold protein of any one of claims 1 to 67 or the fusion protein of any one of claims 68 to 72 and a therapeutic partner.

132. 132. The composition of claim 131 , wherein said therapeutic partner is attached to said engineered scaffold protein or said fusion protein, and said therapeutic partner is covalently bound, non-covalently bound, fused, conjugated, and / or linked to said engineered scaffold protein or said fusion protein.

133. 133. The composition of claim 131 or 132, wherein the therapeutic partner comprises one or more of an anti-cancer agent, an anti-inflammatory agent, an antibacterial agent, an antiviral agent, a cytokine, a toxin, an enzyme, a neuroprotective agent, a soluble factor scavenger, or any combination thereof.

134. 134. The composition of claim 132 or 133, wherein the therapeutic partner can be separated from the engineered scaffold protein or the fusion protein upon binding to the target peptide.

135. 135. The composition of any one of claims 131 to 134, wherein the engineered scaffold protein or the fusion protein may be tissue- or cell-specific.

136. 135. The composition of claim 134, wherein said therapeutic partner is capable of separating from said engineered scaffold protein or said fusion protein upon reaching a specific cell or tissue.

137. 137. A method for treating a disease or disorder, comprising administering to a subject in need thereof a composition according to any one of claims 131 to 136.

138. 1. A method of chimeric antigen receptor (CAR) T cell therapy comprising administering to a subject in need thereof: a. the engineered scaffold protein of any one of claims 1 to 67, or b. A fusion protein according to any one of claims 68 to 72. The method comprises administering

Images